Uses of oxygenated cholesterol sulfates (OCS)

ABSTRACT

Methods of preventing and/or treating ischemia, organ dysfunction and/or organ failure, including multiple organ dysfunction syndrome (MODS), and necrosis and apoptosis associated with organ dysfunction/failure, are provided. For instance, the methods involve contacting organ(s) with an oxygenated cholesterol sulfate (OCS), e.g. 5-cholesten-3,25-diol, 3-sulfate (25HC3S). The organ(s) may be in vivo (e.g. in a patient that is treated with the OCS) or ex vivo (e.g. an organ that has been harvested from a donor and is to be transplanted).

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims priority benefit to U.S. ProvisionalApplication No. 61/920,617, filed Dec. 24, 2013, which application isincorporated herein by reference in its entirety.

This invention was made with government support under a Veteran'sAdministration Merit Review Research Career Scientist Award. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present disclosure generally relates to the prevention and/ortreatment of ischemia, organ dysfunction and/or organ failure, andnecrosis and/or apoptosis associated with organ dysfunction/failure. Forinstance, the present disclosure provides compositions and methods toprevent/treat dysfunction and/or failure of an organ by contacting theorgan with one or more oxygenated cholesterol sulfates (OCS). The organmay be in vivo or ex vivo.

INTRODUCTION

Necrosis is a form of cell injury that results in the premature death ofcells in living tissue by autolysis. Necrosis is caused by factorsexternal to the cell or tissue, such as infection, toxins, or trauma,which result in the unregulated digestion of cell components. Incontrast, apoptosis is a naturally occurring programmed and targetedcause of cellular death. While apoptosis often provides beneficialeffects to the organism, necrosis is almost always detrimental and canbe fatal. In some instances, the two are associated in that necroticcells release factors that elicit apoptosis in surrounding cells andtissues.

Cells that die due to necrosis do not follow the apoptotic signaltransduction pathway but rather various receptors are activated thatresult in the loss of cell membrane integrity and an uncontrolledrelease of products of cell death into the intracellular space. Thisinitiates in the surrounding tissue an inflammatory response whichprevents nearby phagocytes from locating and eliminating the dead cellsby phagocytosis. For this reason, it is often necessary to removenecrotic tissue surgically, a procedure known as debridement. Untreatednecrosis results in a build-up of decomposing dead tissue and celldebris at or near the site of the cell death. A classic example isgangrene.

Organ dysfunction is a condition in which an organ does not perform itsexpected, desired or usual function. Organ failure is organ dysfunctionto such a degree that normal homeostasis cannot be maintained withoutexternal clinical intervention. These two conditions occur on acontinuum of incremental degrees of physiologic derangement and varywidely from a mild degree of organ dysfunction to completelyirreversible organ failure. Organ dysfunction and failure may be acute,developing rapidly (e.g. as a result of acute insult such as a bacterialinfection, severe burns, etc), or may be chronic, developing over a longperiod or time (e.g. as a result of long-term exposure to an organ-toxicmedication). Multiple organ dysfunction syndrome (MODS, previously knownas multiple organ failure (MOF) or multisystem organ failure (MSOF)),refers to the failure of two or more organs or organ systems at the sametime, for example, the cardiovascular and renal systems. In some cases,a single etiological agent or event can be identified as initiating thedisease process but this is not always the case; dysfunction and failuremay be caused by multiple factors and/or the causative agent(s) maynever be identified. A frequent proximal cause is ischemia followed byinflammation and necrosis.

Organ dysfunction and failure have major clinical and economic impacts.The cost of clinical intervention is extremely high and typicallyinvolves intensive life support measures for both acute and chronicdisease. In general, mortality ranges from about 30% to about 100% andhas not changed significantly since the 1980s. The chance of survivaldiminishes as the number of organs involved increases, especially ifcardiovascular dysfunction is involved. For patients that do survive, afull recovery of normal function may not occur for many years, or maynot ever occur.

At present there is no agent available that can reverse establishedorgan failure and therapy is limited to treating the root cause, ifknown, and supportive care such as safeguarding hemodynamics, fluidlevels, pH balance and respiration.

One possible treatment for severe organ failure is the transplantationof an organ from a donor. However, organs that are harvested fortransplant can also suffer from dysfunction due to ischemia fluid loss,pH changes, ketoacidosis and other problems associated with removal fromthe donor and exposure to the ex vivo environment during transport andstorage. For instance, high levels of inflammatory cytokines may bepresent in organs prior to transplant and may cause damage duringtransport and storage. Even though care may be taken to preserve organfunction e.g. by bathing the organ in a specialized fluid duringtransportation, the preservation of viability is still a majorchallenge, and alternative and/or improved agents that can maintain theviability of harvested organs are needed. It would be especiallyadvantageous to have available an agent that is fully biologicallycompatible with donated organs and the bodies of transplant recipients.

There is an urgent need for agents and methods to prevent and treat thedysfunction and/or failure of organs and organ systems, includingprevention and treatment of underlying causes and/or symptoms of organdysfunction and failure, such as sepsis, ischemia, unwanted inflammationand cell death.

SUMMARY

The present disclosure provides a variety of uses for oxygenatedcholesterol sulfates (OCS), including methods of preventing and/ortreating ischemia (e.g., from surgery), necrosis, apoptosis, organdysfunction, and/or organ failure for in vivo and ex vivo organs. Themethods include contacting an organ of interest with at least oneoxygenated cholesterol sulfate (OCS). If the organ of interest is withina patient (in vivo), then contact generally involves administering to apatient harboring the organ an amount of at least one OCS that iseffective or sufficient to prevent and/or treat dysfunction and/orfailure of the organ. Advantageously, the at least one OCS has beenfound to be highly bioavailable, even when administered orally. If theorgan has already been harvested from a subject (i.e. from a donor),and/or is being prepared for harvest from a donor, then contactgenerally involves applying at least one OCS to the organ.

In addition, the present disclosure provides methods of preventingand/or treating diseases and conditions which lead to and/or cause, orare otherwise associated with, organ dysfunction/failure in a patient inneed thereof, by administering to the patient an amount of at least oneOCS that is effective or sufficient to prevent and/or treat the diseaseor condition.

Aspects of the disclosure provide methods of prophylactically treatingor treating ischemia caused by surgery in a subject in need thereof,comprising administering to the subject an amount of5-cholesten-3,25-diol, 3-sulfate (25HC3S) that is sufficient toprophylactically treat or treat ischemia. In some aspects, the ischemiacomprises at least one member selected from cardiac ischemia, brainischemia, bowel ischemia, limb ischemia, and cutaneous ischemia. Inother aspects, the prophylactically treating or treating ischemiacomprises reducing one or more of inflammation, tissue necrosis, organnecrosis, risk of stroke, and reperfusion injury in the subject. Inadditional aspects, the surgery comprises at least one of cardiovascularsurgery, heart surgery, and aneurysm surgery. In further aspects, the25HC3S is administered for not more than seven days prior to surgery,for example on at least a daily basis starting not more than seven daysprior to the surgery. In other aspects, the 25HC3S is administeredduring the surgery. In yet other aspects, the 25HC3S is administered fornot more than seven days after the surgery, for example on at least adaily basis for not more than seven days after the surgery. In someaspects, the surgery is not liver surgery. In other aspects, the surgeryis not a transplant surgery.

Aspects of the disclosure also provide methods of preventing or treatingdysfunction or failure of one or more organs or organ systems in asubject in need thereof, comprising administering to the subject anamount of 5-cholesten-3,25-diol, 3-sulfate (25HC3S) that is sufficientto prevent or treat the dysfunction or failure of the organ or organsystem, wherein if the one or more organs comprises a liver, theadministering occurs for not more than 14 days (2 weeks). In someaspects, the one or more organs comprises at least one member selectedfrom the liver, the kidney, the heart, the brain, and the pancreas. Inadditional aspects, the dysfunction or failure is caused byacetaminophen (ATMP). In further aspects, the 25HC3S is administeredwithin one week of administration of the ATMP. In yet other aspects, thedysfunction or failure is Multiple Organ Dysfunction Syndrome (MODS).

Further aspects of the disclosure provide methods of transplanting oneof more cells, organs or tissues comprising i) removing the one or moreof cells, organs or tissues from a donor, ii) contacting the one or moreof cells, organs or tissues with an amount of 5-cholesten-3,25-diol,3-sulfate (25HC3S) that is sufficient to preserve the one of more cells,organs or tissues; and iii) transplanting the one or more of cells,organs or tissues into a recipient. In further aspects, the one or moreof cells, organs or tissues is not a liver cell, a liver organ or livertissue.

Additional aspects of the disclosure provide methods of preserving an exvivo cell, organ or tissue, comprising contacting the ex vivo cell,organ or tissue, with an amount of 5-cholesten-3,25-diol, 3-sulfate(25HC3S) that is sufficient to preserve the cell, organ or tissue.

Further aspects of the disclosure provide methods of preventing ortreating acute liver failure and/or kidney failure in a subject in needthereof, comprising administering to the subject an amount of5-cholesten-3,25-diol, 3-sulfate (25HC3S) that is effective inpreventing or treating the acute liver failure and/or kidney failure;wherein the acute liver failure and/or kidney failure is caused byacetaminophen (ATMP).

Further aspects of the disclosure:

1. A method of preventing the death of an ex vivo cell, comprising

contacting the ex vivo cell with an amount of 5-cholesten-3,25-diol,3-sulfate (25HC3S) that is sufficient to prevent the death of the cell.

2. The method of 1, wherein the cell is undergoing apoptosis ornecrosis.

3. A method of preventing the death of a cell in a patient, comprising

administering to the subject an amount of 5-cholesten-3,25-diol,3-sulfate (25HC3S) that is sufficient to prevent the death of the cell.

4. The method of 3, wherein the cell is undergoing apoptosis ornecrosis.

5. A method of prophylactically treating or treating ischemia in asubject in need thereof, comprising

administering to the subject an amount of 5-cholesten-3,25-diol,3-sulfate (25HC3S) that is sufficient to prophylactically treat or treatischemia.

6. The method of 5, wherein the ischemia comprises at least one memberselected from cardiac ischemia, brain ischemia, bowel ischemia, limbischemia, and cutaneous ischemia.

7. The method of 5 or 6, wherein the prophylactically treating ortreating comprises reducing at least one of inflammation, tissuenecrosis, organ necrosis, stroke, and reperfusion injury in the subject.

8. The method of any one of 5 to 7, wherein the ischemia is caused bysurgery.

9. The method of 8, wherein the surgery comprises at least one ofcardiovascular surgery, heart surgery, and aneurysm surgery.

10. The method of 8 or 9, wherein the 25HC3S is administered for notmore than seven days prior to the surgery.

11. The method of any one of 8 to 10, wherein the 25HC3S is administeredduring the surgery.

12. The method of any one of 8 to 11, wherein the 25HC3S is administeredfor not more than seven days after the surgery.

13. The method of any one of 8 to 12, wherein the surgery is not liversurgery.

14. The method of any one of 8 to 13, wherein the surgery is not atransplant surgery.

15. The method of any one of 5 to 14, wherein the 25HC3S is administeredto the subject at a dose ranging from about 0.001 mg/kg/day to about 100mg/kg/day.

16. A method of prophylactically treating or treating ischemia caused bysurgery in a subject in need thereof, comprising

administering to the subject an amount of 5-cholesten-3,25-diol,3-sulfate (25HC3S) that is sufficient to prophylactically treat or treatischemia.

17. The method of 16, wherein the ischemia comprises at least one memberselected from cardiac ischemia, brain ischemia, bowel ischemia, limbischemia, and cutaneous ischemia.

18. The method of 16 or 17, wherein the prophylactically treating ortreating comprises reduction in one or more of inflammation, tissuenecrosis, organ necrosis, risk of stroke, and reperfusion injury in thesubject.

19. The method of any one of 16 to 18, wherein the surgery comprises atleast one of cardiovascular surgery, heart surgery, and aneurysmsurgery.

20. The method of any one of 16 to 19, wherein the 25HC3S isadministered for not more than seven days prior to the surgery.

21. The method of any one of 16 to 20, wherein the 25HC3S isadministered during the surgery.

22. The method of any one of 16 to 21, wherein the 25HC3S isadministered for not more than seven days after the surgery.

23. The method of any one of 16 to 22, wherein the surgery is not liversurgery.

24. The method of any one of 16 to 23, wherein the surgery is not atransplant surgery.

25. The method of any one of 16 to 24, wherein the 25HC3S isadministered to the subject at a dose ranging from about 0.001 mg/kg/dayto about 100 mg/kg/day.

26. A method of preventing or treating necrosis of cells, tissues and/ororgans in a subject in need thereof, comprising

administering to the subject an amount of 5-cholesten-3,25-diol,3-sulfate (25HC3S) that is sufficient to prevent or treat the necrosisof cells, tissues and/or organs.

27. The method of 26, wherein the cells, tissues and/or organs compriseat least one member selected from the liver, the kidney, the heart, thebrain, and the pancreas.

28. A method of preventing the spread of necrosis within a tissue ororgan comprising necrotic cells, comprising

administering to the tissue or organ an amount of 5-cholesten-3,25-diol,3-sulfate (25HC3S) that is sufficient to prevent the spread of necrosiswithin the tissue or organ.

29. A method of preventing apoptosis of a cell, comprising

contacting the cell with an amount of 5-cholesten-3,25-diol, 3-sulfate(25HC3S) that is effective in preventing apoptosis of the cell.

30. A method of minimizing apoptosis of cells in a tissue or organ,comprising

contacting the cells with an amount of 5-cholesten-3,25-diol, 3-sulfate(25HC3S) that is sufficient to minimize apoptosis of the cells in thetissue or organ.

31. A method of preventing or treating dysfunction or failure of one ormore organs or organ systems in a subject in need thereof, comprising

administering to the subject an amount of 5-cholesten-3,25-diol,3-sulfate (25HC3S) that is sufficient to prevent or treat thedysfunction or failure of the organ or organ system,

wherein if the one or more organs comprises a liver, the administeringoccurs for not more than 14 days.

32. The method of 31, wherein the one or more organs comprises at leastone member selected from the liver, the kidney, the heart, the brain,and the pancreas.

33. The method of 31 or 32, wherein the dysfunction or failure is causedby acetaminophen (ATMP).

34. The method of 33, wherein the 25HC3S is administered within one weekof administration of the ATMP.

35. The method of any one of 31 to 34, wherein the dysfunction orfailure is Multiple Organ Dysfunction Syndrome (MODS).

36. The method any one of 31 to 35, wherein the 25HC3S is administeredat a dose ranging from about 0.001 mg/kg/day to about 100 mg/kg/day.

37. A method of preventing or treating acute liver failure and/or acutekidney failure in a subject in need thereof, comprising

administering to the subject an amount of 5-cholesten-3,25-diol,3-sulfate (25HC3S) that is effective in preventing or treating the acuteliver failure and/or kidney failure.

38. The method of 37, wherein the acute liver failure and/or acutekidney failure is caused by acetaminophen (ATMP).

39. The method of 37 or 38, wherein the 25HC3S is administered withinone day of onset of the acute liver failure and/or acute kidney failure.

40. The method of any one of 37 to 39, wherein the 25HC3S isadministered for up to 2 weeks after diagnosis of the acute liverfailure and/or acute kidney failure.

41. The method of any one of 37 to 40, wherein the 25HC3S isadministered to the subject at a dose ranging from about 0.001 mg/kg/dayto about 100 mg/kg/day.

42. A method of decreasing a risk of mortality in a subject experiencingor at risk of experiencing dysfunction or failure of an organ or organsystem, comprising

administering to the subject an amount of 5-cholesten-3,25-diol,3-sulfate (25HC3S) that is sufficient to decrease the risk of mortality.

43. A method of preserving an ex vivo cell, organ or tissue, comprising

contacting the ex vivo cell, organ or tissue, with an amount of5-cholesten-3, 25-diol, 3-sulfate (25HC3S) that is sufficient topreserve the cell, organ or tissue.

44. A method of transplanting one of more cells, organs or tissuescomprising

removing the one or more of cells, organs or tissues from a donor,

contacting the one or more of cells, organs or tissues with an amount of5-cholesten-3,25-diol, 3-sulfate (25HC3S) that is sufficient to preservethe one of more cells, organs or tissues; and

transplanting the one or more of cells, organs or tissues into arecipient.

45. The method of 44, wherein the one or more of cells, organs ortissues is not a liver cell, a liver organ or liver tissue.

46. A composition comprising

an ex vivo cell, organ or tissue and

5-cholesten-3,25-diol, 3-sulfate (25HC3S).

47. The composition of 46, further comprising an oxygenatedphysiologically compatible carrier medium.

48. A composition comprising:

an active agent comprising at least one member selected from ibuprofen,aspirin, and acetaminophen; and

5-cholesten-3,25-diol, 3-sulfate (25HC3S).

49. A method of prophylactically treating or treating sepsis in asubject in need thereof, comprising

administering to the subject an amount of 5-cholesten-3,25-diol,3-sulfate (25HC3S) that is sufficient to prophylactically treat or treatthe sepsis.

50. The method of 49, wherein the prophylactically treating or treatingsepsis comprises prophylactically treating or treating damage associatedwith sepsis, wherein the damage is optionally dysfunction or failure ofone or more organs.

51. The method of 50, wherein the one or more organs comprises at leastone member selected from the liver, the kidney, the heart, the brain,and the pancreas.

52. A method of preventing or treating necrosis and/or apoptosisassociated with necrosis of cells or tissue in a subject in needthereof, comprising

administering to the subject an amount of one or both of5-cholesten-3,25-diol, 3-sulfate (25HC3S) and 5-cholesten 3, 25-diol,disulfate (25HCDS) that is effective in preventing or treating thenecrosis and/or apoptosis.

53. The method of 52, wherein the tissue is liver tissue and/or kidneytissue.

54. The method of 53, wherein the necrosis is caused by acetaminophen(ATMP).

55. A method of preventing or treating acute liver failure and/or kidneyfailure in a subject in need thereof, comprising

administering to the subject an amount of one or both of5-cholesten-3,25-diol, 3-sulfate (25HC3S) and 5-cholesten 3, 25-diol,disulfate (25HCDS) that is effective in preventing or treating the acuteliver failure and/or kidney failure.

56. The method of 55, wherein the acute liver failure and/or kidneyfailure is caused by acetaminophen (ATMP).

57. The method of any of 3, 4, 26 to 28, 42, and 49 to 56, wherein the25HC3S is administered at a dose ranging from about 0.001 mg/kg/day toabout 100 mg/kg/day.

58. The method of any of 3 to 28, 31 to 42, and 49 to 57, wherein theadministering is performed orally or by injection.

59. The method of any of 3 to 28, 31 to 42, and 49 to 57, wherein theadministering is performed from once to 3 times per day.

60. 5-cholesten-3,25-diol, 3-sulfate (25HC3S) for use in a method ofmedical treatment that comprises preventing the death of a cell.

61. A method of preventing the death of a cell ex vivo, comprising

contacting the cell with a an amount of 5-cholesten-3,25-diol, 3-sulfate(25HC3S) that is sufficient to prevent the death of the cell.

62. 5-cholesten-3,25-diol, 3-sulfate (25HC3S) for use in a method ofprophylactically treating or treating ischemia.

63. 5-cholesten-3,25-diol, 3-sulfate (25HC3S) for use in a method ofprophylactically treating or treating ischemia caused by surgery.

64. 5-cholesten-3,25-diol, 3-sulfate (25HC3S) for use in a method ofpreventing or treating necrosis of cells, tissues and/or organs in asubject in need thereof.

65. 5-cholesten-3,25-diol, 3-sulfate (25HC3S) for use in a method ofmedical treatment that comprises preventing the spread of necrosiswithin a tissue or organ comprising necrotic cells.

66. 5-cholesten-3,25-diol, 3-sulfate (25HC3S) for use in a method ofmedical treatment that comprises preventing apoptosis of a cell.

67. 5-cholesten-3,25-diol, 3-sulfate (25HC3S) for use in a method ofmedical treatment that comprises minimizing apoptosis of cells in atissue or organ.

68. 5-cholesten-3,25-diol, 3-sulfate (25HC3S) for use in a method ofpreventing or treating dysfunction or failure of one or more organs ororgan systems in a subject in need thereof, wherein if the one or moreorgans comprises a liver the method comprises administering the5-cholesten-3,25-diol, 3-sulfate (25HC3S) for no more than 14 days.69. 5-cholesten-3,25-diol, 3-sulfate (25HC3S) for use in a method ofpreventing or treating acute liver failure and/or acute kidney failurein a subject in need thereof.70. 5-cholesten-3,25-diol, 3-sulfate (25HC3S) for use of 67, wherein theacute liver failure and/or acute kidney failure is caused byacetaminophen (ATMP).71. 5-cholesten-3,25-diol, 3-sulfate (25HC3S) for use in a method ofdecreasing a risk of mortality in a subject experiencing or at risk ofexperiencing dysfunction or failure of an organ or organ system.72. 5-cholesten-3,25-diol, 3-sulfate (25HC3S) for use in a method oftreatment comprising: removing, optionally by surgery, one or more ofcells, organs or tissues from a donor; and contacting, ex vivo, the oneor more of cells, organs or tissues with an amount of5-cholesten-3,25-diol, 3-sulfate (25HC3S) that is sufficient to preservethe one of more cells, organs or tissues.73. 5-cholesten-3,25-diol, 3-sulfate (25HC3S) for use in a method oftreatment comprising: contacting, ex vivo, one or more of cells, organsor tissues with an amount of 5-cholesten-3,25-diol, 3-sulfate (25HC3S)that is sufficient to preserve the one of more cells, organs or tissues;and transplanting, optionally by surgery, the one or more of cells,organs or tissues into a recipient.74. 5-cholesten-3,25-diol, 3-sulfate (25HC3S) for use in a method oftreatment comprising: removing, optionally by surgery, one or more ofcells, organs or tissues from a donor; contacting, ex vivo, the one ormore of cells, organs or tissues with an amount of5-cholesten-3,25-diol, 3-sulfate (25HC3S) that is sufficient to preservethe one of more cells, organs or tissues; and transplanting, optionallyby surgery, the one or more of cells, organs or tissues into arecipient.75. 5-cholesten-3,25-diol, 3-sulfate (25HC3S) for use in a method ofprophylactically treating or treating sepsis.76. 5-cholesten-3,25-diol, 3-sulfate (25HC3S) and/or 5-cholesten 3,25-diol, disulfate (25HCDS) for use in a method of preventing ortreating necrosis and/or apoptosis associated with necrosis of cells ortissue in a subject in need thereof.77. 5-cholesten-3,25-diol, 3-sulfate (25HC3S) and/or 5-cholesten 3,25-diol, disulfate (25HCDS) for use in a method of preventing ortreating acute liver failure and/or kidney failure in a subject in needthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-D. Effect of 25HC3S on recovery of acute liver failure inducedby acetaminophen. Normal represents sera from normal control mice; ATMP,from mice treated with acetaminophen and vesicle injection; ATMP+25HC3S,from mice with acetaminophen and 25HC3S injection. A, ALT representsalanine aminotransferase; B, AST aspartate aminotransferase; C, ALK,alkaline phosphatase; D, LDH, lactate dehydrogenase. Each valuerepresents mean of two animals.

FIGS. 2A and B. Effect of 25HC3S on recovery after administration ofacetaminophen (ATMP). Normal represents sera from normal control mice;ATMP, mice treated with ATMP and vehicle; ATMP+25HC3S, mice treated withATMP and 25HC3S. A, enzyme activities: ALT, alanine aminotransferase;AST, aspartate aminotransferase; ALK, alkaline phosphatase; LDH, lactatedehydrogenase; B, serum concentrations of BUN (blood urea nitrogen) andglucose. Each value represents mean of two animals.

FIGS. 3A and B. Concentration of 25HC3S in A, blood and B, the indicatedtissues of rats subjected to high levels of ATMP.

FIG. 4. Mortality data of control and 25HC3S treated rats at days 1, 2,3, 4, and 10 days after liver ischemia.

FIG. 5A-D. Relative enzyme activity (units per deciliter of blood) ofcontrol vs treated rats for A, alanine aminotransferase (ALT), B,aspartate aminotransferase (AST), C, alkaline phosphatase (AKP) and D,antidiuretic hormone (ADH). Control rats received vehicle; treated ratsreceived 25HC3S.

FIGS. 6A and B. Serum creatine and BUN values for renalischemia/reperfusion experiment. A, serum creatine levels as apercentage of vehicle; B, serum BUN levels as a percentage of vehicle.

FIG. 7. 24 hour survival rates for heart ischemia/reperfusionexperiment.

FIG. 8A-L. Results after brain ischemia injury. A, 7-point neuroscore;B, 20 point neuroscore; C, limb placing; D, 24-hr lesion volume (mm³);E, 7-day lesion volume (mm³); F, 24-hr oedema volume (mm³); G, 7-dayoedema volume (mm³); H, 24-hr lesion volume (%); I, 7-day oedema volume(%); J, 24-hr T2 lesion (ms); K, 7-day T2 lesion (ms); L, body weights(vehicle/sham, vehicle/stroke and vehicle/25HC3S).

FIG. 9A-C. Sepsis studies. Mice were intravenously injected with twodifferent concentrations of lipopolysaccharide and then with vehicle or25 HC3S. A, 40 mg/kgLPS; B, 30 mg/kg LPS; C, 4 mg/ml LPS.

FIG. 10. Level of 25HC3S in plasma samples from Phase I cohorts 1-4.

FIG. 11. Schematic representation illustrating timing (days) of blooddraws, surgery, treatment and sacrifice of animals.

DETAILED DESCRIPTION OF THE INVENTION

Methods for preventing and/or treating organ or organ system dysfunctionand/or failure are described herein, as are methods of treating unwantednecrosis and/or apoptosis associated with organ failure. The methodsinclude contacting an organ of interest with at least one oxygenatedcholesterol sulfate (OCS). If the organ of interest is within a patient(in vivo), then contact generally involves administering to the patientan amount of at least one OCS that is effective or sufficient to preventand/or treat dysfunction and/or failure of one or more organs or organsystems in the patient, e.g. is sufficient to prevent or treat at leastone symptom of organ dysfunction or failure exhibited by the patient. Ifan organ has already been harvested from a subject (i.e. from a donor),and is thus ex vivo, then contact generally involves contacting theorgan with at least one OCS, i.e. applying at least one OCS to theorgan, to preserve the organ, i.e. maintain the viability of the organ,and/or enhance maintenance of the organ, until it is transplanted.

Methods of preventing and/or treating conditions which lead to, cause orare caused by, or which are associated with organ dysfunction andfailure are also described, e.g. prevention and/or treatment ofinflammation, cell death (e.g. necrosis), consequences of ischemia,sepsis, and others. The methods involve administering, to a subject inneed thereof, an amount of at least one OCS that is effective orsufficient to prevent and/or treat the condition.

In some aspects, the populations of subjects treated by the methodsdescribed herein may or may not have symptoms of and/or been diagnosedwith high levels of cholesterol (hypercholesterolemia, e.g. cholesterollevels in serum in the range of about 200 mg/dl or more), or with acondition associated with high levels of cholesterol e.g.hyperlipidemia, atherosclerosis, heart disease, stroke, Alzheimer's,gallstone diseases, cholestatic liver diseases, etc. In some aspects,the populations of subjects treated by the methods described herein donot have symptoms of and/or have not been diagnosed with high levels ofcholesterol (hypercholesterolemia, e.g. cholesterol levels in serum inthe range of about 200 mg/dl or more), or with a condition associatedwith high levels of cholesterol e.g. hyperlipidemia, atherosclerosis,heart disease, stroke, Alzheimer's, gallstone diseases, cholestaticliver diseases, etc.

In further aspects, the populations of subjects treated by the methodsdescribed herein may or may not have symptoms of and/or been diagnosedwith liver disorders such as hepatitis, inflammation of the liver,caused mainly by various viruses but also by some poisons (e.g.alcohol); autoimmunity (autoimmune hepatitis) or hereditary conditions;non-alcoholic fatty liver disease, a spectrum in disease, associatedwith obesity and characterized by an abundance of fat in the liver,which may lead to hepatitis, i.e. steatohepatitis and/or cirrhosis;cirrhosis, i.e. the formation of fibrous scar tissue in the liver due toreplacing dead liver cells (the death of liver cells can be caused, e.g.by viral hepatitis, alcoholism or contact with other liver-toxicchemicals); haemochromatosis, a hereditary disease causing theaccumulation of iron in the body, eventually leading to liver damage;cancer of the liver (e.g. primary hepatocellular carcinoma orcholangiocarcinoma and metastatic cancers, usually from other parts ofthe gastrointestinal tract); Wilson's disease, a hereditary diseasewhich causes the body to retain copper; primary sclerosing cholangitis,an inflammatory disease of the bile duct, likely autoimmune in nature;primary biliary cirrhosis, an autoimmune disease of small bile ducts;Budd-Chiari syndrome (obstruction of the hepatic vein); Gilbert'ssyndrome, a genetic disorder of bilirubin metabolism, found in about 5%of the population; glycogen storage disease type II; as well as variouspediatric liver diseases, e.g. including biliary atresia, alpha-1antitrypsin deficiency, alagille syndrome, and progressive familialintrahepatic cholestasis, etc. In addition, liver damage from trauma mayalso be treated, e.g. damage caused by accidents, gunshot wounds, etc.Further, liver damage caused by certain medications may be prevented ortreated, for example, drugs such as the antiarrhythmic agent amiodarone,various antiviral drugs (e.g. nucleoside analogues), aspirin (rarely aspart of Reye's syndrome in children), corticosteroids, methotrexate,tamoxifen, tetracycline, etc. are known to cause liver damage. Infurther aspects, the populations of subjects treated by the methodsdescribed herein do not have symptoms of and/or have not been diagnosedwith liver disorders such as hepatitis, inflammation of the liver,caused mainly by various viruses but also by some poisons (e.g.alcohol); autoimmunity (autoimmune hepatitis) or hereditary conditions;non-alcoholic fatty liver disease, a spectrum in disease, associatedwith obesity and characterized by an abundance of fat in the liver,which may lead to hepatitis, i.e. steatohepatitis and/or cirrhosis;cirrhosis, i.e. the formation of fibrous scar tissue in the liver due toreplacing dead liver cells (the death of liver cells can be caused, e.g.by viral hepatitis, alcoholism or contact with other liver-toxicchemicals); haemochromatosis, a hereditary disease causing theaccumulation of iron in the body, eventually leading to liver damage;cancer of the liver (e.g. primary hepatocellular carcinoma orcholangiocarcinoma and metastatic cancers, usually from other parts ofthe gastrointestinal tract); Wilson's disease, a hereditary diseasewhich causes the body to retain copper; primary sclerosing cholangitis,an inflammatory disease of the bile duct, likely autoimmune in nature;primary biliary cirrhosis, an autoimmune disease of small bile ducts;Budd-Chiari syndrome (obstruction of the hepatic vein); Gilbert'ssyndrome, a genetic disorder of bilirubin metabolism, found in about 5%of the population; glycogen storage disease type II; as well as variouspediatric liver diseases, e.g. including biliary atresia, alpha-1antitrypsin deficiency, alagille syndrome, and progressive familialintrahepatic cholestasis, etc. In addition, liver damage from trauma mayalso be treated, e.g. damage caused by accidents, gunshot wounds, etc.Further, liver damage caused by certain medications may be prevented ortreated, for example, drugs such as the antiarrhythmic agent amiodarone,various antiviral drugs (e.g. nucleoside analogues), aspirin (rarely aspart of Reye's syndrome in children), corticosteroids, methotrexate,tamoxifen, tetracycline, etc. are known to cause liver damage.

In further aspects, the populations of subjects treated by the methodsdescribed herein may or may not have symptoms of non-alcoholic fattyliver disease (NAFLD) and/or nonalcoholic steatohepatitis (NASH). Infurther aspects, the populations of subjects treated by the methodsdescribed herein do not have symptoms of non-alcoholic fatty liverdisease (NAFLD) and/or nonalcoholic steatohepatitis (NASH).

Definitions

The following definitions are used throughout:

Prevent and Treat

As used herein, “prophylactically treat” (“prophylactic treatment”,“prophylactically treating” etc.) and “prevent” (“prevention”,preventing” etc.) refer to warding off or averting the occurrence of atleast one symptom of a disease or unwanted condition such as organdysfunction or failure, by prophylactic administration of at least oneOCS to a subject in need thereof. Generally, “prophylactic” or“prophylaxis” relates to a reduction in the likelihood of the patientdeveloping a disorder. Typically, the subject is considered by one ofskill in the art to be at risk of or susceptible to developing at leastone symptom of the disease or unwanted condition, or is considered to belikely to develop at least one symptom of the disease/condition in theabsence of medical intervention. Generally, however, for “prevention” or“prophylactic treatment”, administration occurs before the subject has,or is known or confirmed to have, symptoms of the disease (condition,disorder, syndrome, etc.; unless otherwise indicated, these terms areused interchangeably herein). In other words, symptoms may not yet beovert or observable. The subject may be considered at risk due to avariety of factors, including but not limited to: geneticpredisposition; an impending medical or surgical procedure (e.g.surgery, use of a contrast dye in imaging, chemotherapy, etc.); recentcertain or suspected or unavoidable future exposure to a toxic agent(e.g. a toxic chemical or medication, radiation, etc.); or exposure toor experience of another stressor or combination of stressors thatis/are linked to or associated with the development of thedisease/condition which is being prevented. In some aspects of theprevention of organ dysfunction/failure, the subject may already displaysymptoms of a potential precursor of organ dysfunction/failure, forexample, ischemic, sepsis, a harmful or inappropriate level ofinflammation, deleterious cell death, necrosis, etc. In such aspects,treatment of the subject may prevent the noxious or harmful effects oroutcomes (results) of the precursor condition. “Prevention” or“prophylactic treatment” of a disease or condition may involvecompletely preventing the occurrence of detectable symptoms, or,alternatively, may involve lessening or attenuating the degree, severityor duration of at least one symptom of the disease that would occur inthe absence of the medical interventions provided herein, i.e. unlessone or more OCSs is administered. Alternatively, the subject may beexperiencing early stage symptoms and what is prevented is theprogression to full-blown disease.

In some aspects, the disease outcome or result that is prevented isdeath of the subject.

“Treat” (treatment, treating, etc.) as used herein refers toadministering at least one OCS to a subject that already exhibits atleast one symptom of the disease. In other words, at least one parameterthat is known to be associated with the disease has been measured,detected or observed in the subject. Organ dysfunction/failure and/orprecursors thereof that are treated as described herein are caused bysomewhat predictable factors (e.g. see the above description of diseasesand conditions which may lead to organ dysfunction/failure), or byunexpected causes such as trauma due to accidents (recreational andnon-recreational), war, undiagnosed allergies or other risk factors,etc. “Treatment” of a disease involves the lessening or attenuation, orin some instances, the complete eradication, of at least one symptom ofthe disease that was present prior to or at the time of administrationof one or more OCSs. Thus, for example, treatment of ischemia includespreventing or treating damage associated with ischemia and, for exampletreatment of sepsis includes preventing or treating damage associatedwith sepsis.

Those of skill in the art will recognize that one or more of organdysfunction, organ failure, and/or one or more conditions which areprecursors of organ dysfunction or failure may be comorbid, i.e. may bepresent in a subject or individual at the same time. For example, asubject may have active sepsis that results in organ failure. Thus,preventing and/or treating may overlap in that treating sepsis may, atthe same time, prevent the occurrence of organ failure; or treatingischemia may prevent or treat inflammation that occurs following anischemic event, that would lead to organ failure but for theadministration of an OCS.

Examples of OCS that are used in the methods and compositions describedherein include but are not limited to 5-cholesten-3,25-diol, 3-sulfate(25HC3S); 5-cholesten, 3b,25-diol, disulfate (25HCDS); (5-cholestene,3,27-diol, 3-sulfate); (5-cholestene, 3,27-diol, 3,27-disulfate);(5-cholestene, 3,7-diol, 3-sulfate); (5-cholestene, 3,7-diol,3,7-disulfate); (5-cholestene, 3,24-diol, 3-sulfate); (5-cholestene,3,24-diol, 3,24-disulfate); and (5-cholestene, 3-ol, 24,25-epoxy3-sulfate). Disclosure of 25HC3S is found in, e.g., U.S. Pat. No.8,399,441, which is incorporated herein by reference in its entirety.Disclosure of 25HCDS is found, e.g., in WO 2013/154752, which isincorporated by reference in its entirety. In certain aspects, the OCSare selected from 5-cholesten-3,25-diol, 3-sulfate (25HC3S) and5-cholesten, 3b, 25-diol, disulfate (25HCDS) (either alone or incombination). In further aspects, the OCS is 5-cholesten-3, 25-diol,3-sulfate (25HC3S).

The OCSs are typically synthetic versions of OCSs that occur naturallyin the body. The exogenous OCS may be administered forms not naturallyfound in the body, and in concentrations that are significantly higherthan those which occur naturally. For 25HC3S, natural levels typicallyrange from e.g. about 2 ng/ml or less up to about 5 ng/ml. Theconcentration of OCS (e.g. 25HC3S) in the blood or plasma of a patientthat is treated with an OCS (e.g. 25HC3S) is generally greater thanabout 5 ng/ml, and generally ranges from about 50 ng/ml to about 5000ng/ml, such as about 80 ng/ml to about 3000 ng/ml, e.g. from about 100to about 2000 ng/ml, or from about 200 to about 1000 ng/ml.

As used herein, “organ” refers to a differentiated and/or relativelyindependent body structure comprising cells and tissues that performssome specialized function in the body of an organism. An “organ system”refers to two or more organs that work together in the execution of abody function. A hollow organ is an internal visceral organ (viscus)that forms a hollow tube or pouch, or that includes a cavity. Exemplaryorgans, the dysfunction or failure of which are prevented and/or treatedby the administration of or contact with one or more OCS, include butare not limited to: heart, lungs, (e.g., lungs damaged by pulmonaryfibrosis, e.g., associated with chronic asthma), liver, pancreas,kidneys, brain, intestines, colon, thyroid, etc. In some cases, thedysfunction or failure which is prevented and/or treated by theadministration of the one or more OCS involves an organ other than theliver, for example heart, lungs, pancreas, kidneys, brain, intestines,colon, etc. In general, methods and compositions described herein thatrefer to “organs” should also be understood to include “organ systems”,unless otherwise specified.

“Organ dysfunction” denotes a condition or a state of health where anorgan does not perform its expected function. Organ function representsthe expected function of the respective organ within physiologic ranges.The person skilled in the art is aware of the respective function of anorgan during medical examination. Organ dysfunction typically involves aclinical syndrome in which the development of progressive andpotentially reversible physiological dysfunction in an organ, optionallyin the absence of anatomic injuries.

“Organ failure” denotes an organ dysfunction to such a degree thatnormal homeostasis cannot be maintained without external clinicalintervention.

“Acute organ dysfunction” refers to reduced organ function that occursrapidly—in days or weeks (e.g., within 26 weeks, within 13 weeks, within10 weeks, within 5 weeks, within 4 weeks, within 3 weeks, within 2weeks, within 1 week, within 5 days, within 4 days, within 3 days, orwithin 2 days)—usually in a person who has no pre-existing disease.

“Acute organ failure” refers to loss of organ function that occursrapidly—in days or weeks (e.g., within 26 weeks, within 13 weeks, within10 weeks, within 5 weeks, within 4 weeks, within 3 weeks, within 2weeks, within 1 week, within 5 days, within 4 days, within 3 days, orwithin 2 days)—usually in a person who has no pre-existing disease. Forinstance, the term “acute renal failure” means a rapid deterioration inrenal function sufficient to result in accumulation of waste products inthe body. Acute liver failure is discussed in more detail below.

As used herein, “ischemia” refers to a reduction in blood flow to anorgan.

The terms “sepsis” and “septicemia” refer to a morbid conditionresulting from the invasion of the bloodstream by microorganisms andtheir associated endotoxins.

“Endotoxin” refers to any harmful components of microbial cells such aslipopolysaccharides from the Gram-negative bacterial cell wall,peptidoglycans from Gram-positive bacteria, and mannan from fungal cellwalls.

Description of Administration of Oxygenated Cholesterol Sulfates (OCS)

Implementation of the methods generally involves identifying patientssuffering from or at risk of developing organ dysfunction or failure, ora condition associated with organ dysfunction or failure, andadministering one or more OCS in an acceptable form by an appropriateroute. The exact dosage to be administered may vary depending on theage, gender, weight and overall health status of the individual patient,as well as the precise etiology of the disease. However, in general foradministration in mammals (e.g. humans), dosages in the range of fromabout 0.001 to about 100 mg or more of compound per kg of body weightper 24 hr., and preferably about 0.01 to about 50 mg of compound per kgof body weight per 24 hr., and more preferably about 0.1 to about 10 mgof compound per kg of body weight per 24 hr. are effective. Daily dosesgenerally range from about 0.1 milligrams to about 5000 milligrams ofOCS such as 25HC3S (or a pharmaceutically acceptable salt thereof) perperson per day. In some aspects, the dose is from about 10 milligrams toabout 2000 milligrams per person per day, or about 100 milligrams toabout 1000 milligrams per person per day. The dose will vary with theroute of administration, the bioavailability, and the particularformulation that is administered, as well as according to the nature ofthe malady that is being prevented or treated. Further, the effectivedose can vary depending upon factors such as gender, age, and otherconditions of the patient, as well as the extent or progression of thedisease condition being treated.

Administration may be oral or parenteral, including intravenously,intramuscularly, subcutaneously, intradermal injection, intraperitonealinjection, etc., or by other routes (e.g. transdermal, sublingual,rectal and buccal delivery, inhalation of an aerosol, intravaginally,intranasally, topically, as eye drops, via sprays, etc.). The route ofadministration will depend on the nature or the condition that istreated, e.g. on the type or degree of organ injury and/or organ failureand/or associated necrosis and/or apoptosis, and whether the treatmentis prophylactic or intended to effect a cure. For example, to achieve apreventative effect before organ dysfunction has occurred, oral dosingmay be sufficient, especially in view of the excellent bioavailabilityof orally administered OCS. Further, administration of the compound byany means may be carried out as a single mode of therapy, or inconjunction with other therapies and treatment modalities, e.g. dietregimens, etc.

The subject to whom the OCS is administered is generally a mammal,frequently a human, but this is not always the case. Veterinaryapplications of this technology are also contemplated, e.g. forcompanion pets (cats, dogs, etc.), or for livestock and farm animals,for horses, and even for “wild” animals that have special value or thatare under the case of a veterinarian, e.g. animals in preserves or zoos,injured animals that are being rehabilitated, etc.

In some aspects, the compositions are administered in conjunction withother treatment modalities such as various pain relief medications,anti-arthritis agents, various chemotherapeutic agents, antibioticagents, and the like, depending on the malady that is afflicting thesubject. “In conjunction with” refers to both administration of aseparate preparation of the one or more additional agents, and also toinclusion of the one or more additional agents in a composition of thepresent disclosure. In particular, the OCS may be administered inconjunction with an agent that is known to cause organ damage in orderto prevent the organ damage. For example, aspirin, ibuprofen andacetaminophen all have potential serious organ-damaging side effectswhen taken long term, or when taken by certain venerable groups (e.g.the very young, the elderly, etc.), or when overdoses are ingested, etc.Accordingly, dosage forms comprising at least one OCS and one or more ofsuch agents are contemplated.

The amount of OCS that is effective in protecting against aspirin-,ibuprofen- or acetaminophen-induced organ injury can be determined bystandard clinical techniques. In addition, in vitro or in vivo assayscan optionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed will also depend on the route ofadministration, and can be decided according to the judgment of thepractitioner and each patient's circumstances. However, suitable dailydoses for oral administration generally from about 0.1 milligrams toabout 5000 milligrams of OCS such as 25HC3S (or a pharmaceuticallyacceptable salt thereof) per person per day. In some aspects, an oraldose is from about 10 milligrams to about 2000 milligrams per person perday, or about 100 milligrams to about 1000 milligrams per person perday. Oral compositions are generally contemplated for prophylactic use,e.g. when the potentially dangerous agent is taken for a long period oftime (weeks, months or years) and it is desired to prevent organ damageor other adverse effects. However, when treatment is needed for damagethat has already occurred, the compositions are generally formulated forparenteral or, more usually, for intravenous administration.

The compounds may be administered in the pure form or in apharmaceutically acceptable formulation including suitable elixirs,binders, and the like (generally referred to a “carriers”) or aspharmaceutically acceptable salts (e.g. alkali metal salts such assodium, potassium, calcium or lithium salts, ammonium, etc.) or othercomplexes. It should be understood that the pharmaceutically acceptableformulations include liquid and solid materials conventionally utilizedto prepare both injectable dosage forms and solid dosage forms such astablets and capsules and aerosolized dosage forms. In addition, thecompounds may be formulated with aqueous or oil based vehicles. Watermay be used as the carrier for the preparation of compositions (e.g.injectable compositions), which may also include conventional buffersand agents to render the composition isotonic. Other potential additivesand other materials (preferably those which are generally regarded assafe [GRAS]) include: colorants; flavorings; surfactants (TWEEN®, oleicacid, etc.); solvents, stabilizers, elixirs, and binders or encapsulants(lactose, liposomes, etc). Solid diluents and excipients includelactose, starch, conventional disintegrating agents, coatings and thelike. Preservatives such as methyl paraben or benzalkonium chloride mayalso be used. Depending on the formulation, it is expected that theactive component (at least one OCS) will be present as about 1% to about99% of the composition and the vehicular “carrier” will constitute about1% to about 99% of the composition. The pharmaceutical compositions ofthe present disclosure may include any suitable pharmaceuticallyacceptable additives or adjuncts to the extent that they do not hinderor interfere with the therapeutic effect of the OCS(s). Additionalsuitable agents that may be co-administered or co-formulated alsoinclude other agents that are used to e.g. combat acetaminophentoxicity, including but not limited to: metabolites of the methionineand/or glutathione biosynthetic pathways such as S-adenosylhomocysteine(SAH), S-methylmethionine (SMM), cystine, betaine, etc. or various formsand/or salts thereof e.g. acetylcysteine (e.g. intravenousN-acetylcysteine), various neutraceuticals, etc.

The administration of the compound of the present disclosure may beintermittent, or at a gradual or continuous, constant or controlledrate. In addition, the time of day and the number of times per day thatthe pharmaceutical formulation is administered may vary and are bestdetermined by a skilled practitioner such as a physician. For example,the compound may be administered within 1 week, such as within 1 day,within 12 hours, within 1 hour, or within 10 minutes, of an overdosee.g. of an agent that causes organ damage. The compound may beadministered at least once a day (e.g., twice daily) before surgery forat least 1 month or at least 1 week, or at least 1 day before surgery,or even during surgery, e.g. surgery related to or associated with orwhich may cause organ failure (e.g. surgery that involves intentionalischemia/reperfusion). The compound may also be administered on at leasta daily basis (e.g., twice daily) after surgery for at least 1 day, atleast 1 week, or at least 1 month. For example, the surgery may be heartsurgery (e.g., coronary artery bypass grafting (CABG)), cardiovascularsurgery, heart-lung transplant, lung surgery (e.g., pulmonary embolismsurgery), deep vein thrombosis (DVT) surgery, brain surgery, liversurgery, bile duct surgery, kidney surgery (e.g., kidney stone surgery),gastrointestinal surgery (e.g., intestinal, intestinal blockage,diverticulitis, or intestinal torsion surgery), or aneurysm surgery. Insome cases, such as when the one or more organs to be treated comprisesa liver, the administering may occur for not more than 14 days, such asnot more than 10 days, not more than 8 days, not more than 5 days, ornot more than 1 day.

The OCS are typically administered as compositions that are prepared insolid forms such as tablets, pills, powders, suppositories, variousslow- or extended-release formulations, and the like, or as liquidsolutions, suspensions, emulsions, etc. or liquids suitable forinjection and/or intravenous administration. Solid forms suitable forsolution in, or suspension in, liquids prior to administration may alsobe prepared. The active ingredients may be mixed with excipients whichare pharmaceutically acceptable and compatible with the activeingredients, e.g. pharmaceutically and physiologically acceptablecarriers. Suitable excipients include, for example, water, saline,dextrose, glycerol, ethanol and the like, or combinations thereof. Inaddition, the composition may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,and the like. Oral dosage forms may include various thickeners,flavorings, diluents, emulsifiers, dispersing aids, binders, coatingsand the like. The composition of the present disclosure may contain anysuch additional ingredients so as to provide the composition in a formsuitable for the intended route of administration. The final amount ofOCS in a formulation may also vary but in general will be from about1-99%. Still other suitable formulations for use in the presentdisclosure can be found, for example in Remington's PharmaceuticalSciences, Philadelphia, Pa., 19th ed. (1995); and Akers, Michael J.Sterile Drug Products: Formulation, Packaging, Manufacturing andQuality; publisher Informa Healthcare (2010).

The compositions (preparations) of the present disclosure may beformulated for and administered by any of the many suitable means whichare known to those of skill in the art, including but not limited to:orally, by injection, rectally, by inhalation, intravaginally,intranasally, topically, as eye drops, via sprays, etc. In some aspects,the mode of administration is oral, by injection or intravenously.Typically, oral administration is particularly effective when usedprophylactically, e.g. to prevent organ damage (e.g. caused by ornecrosis and/or apoptosis) and that would otherwise occur in a patientwho is taking an organ-damaging agent for a prolonged period of time,e.g. weeks, months or years. When damage has already occurred, andespecially when acute organ failure is diagnosed, the route ofadministration is generally parenteral or intravenous to speed deliveryof the OCS.

Prevention and/or Treatment of Organ and/or Organ System Dysfunctionand/or Failure

In some aspects, the present disclosure provides methods for preventingand/or treating the dysfunction and/or failure of one or more organs ororgan systems in a subject in need thereof. In some aspects, the organand/or organ system dysfunction and/or failure is acute.

The methods may include administering to the subject a therapeuticallyeffective or sufficient amount of one or more OCS. The amount issufficient to prevent and/or treat dysfunction of the organ(s) beingtreated, or to prevent and/or treat failure of the organ(s) beingtreated. In some aspects, the organ failure that is treated is MultipleOrgan Dysfunction Syndrome (MODS). The methods generally includeidentifying or diagnosing subjects who are in need of such treatment,e.g. subjects that would benefit from such treatment e.g. due to beingsusceptible to organ dysfunction or failure, or already exhibiting atleast one sign or symptom of organ dysfunction or failure. For example,the subject may be a member of a particular patient population such asthose with disease resulting from acute insult (acute organ injuryresulting from bacterial infection, severe burns, trauma, etc), orchronic conditions (long-term exposure to organ-damaging medication),and/or from other causes which are discussed in more detail below.

The patient group(s) addressed by the present disclosure can also bedefined as follows. The SOFA system was created in a consensus meetingof the European Society of Intensive Care Medicine in 1994 and furtherrevised in 1996. The SOFA is a six-organ dysfunction/failure scoremeasuring multiple organ failure daily. Each organ is graded from 0(normal) to 4 (the most abnormal), providing a daily score of 0 to 24points. The objective of the SOFA is to create a simple, reliable, andcontinuous score for clinical staff Sequential assessment of organdysfunction during the first few days of intensive care unit (ICU) orhospital admission is a good indicator of prognosis. Both the mean andhighest SOFA scores are particularly useful predictors of outcome.

In a specific aspect, the patient group pursuant to the invention is onehaving as a lower threshold at least one SOFA score, being at 1 for oneof the clinical criteria of respiration, or liver, or coagulation, orcardiovascular, or CNS, or renal on the day of admission to hospital orIntensive Care Unit (ICU). Thus, said patient group is in need oftherapeutic intervention pursuant to the present invention, and thus inneed for prevention or reduction of organ dysfunction or organ failure.

In another specific aspect, the patient group pursuant to the presentdisclosure is one having as lower threshold at least two SOFA scores,being at 1 each for two of the clinical criteria respiration, and/orliver, and/or coagulation, and/or cardiovascular, and/or CNS, and/orrenal on the day of admission to hospital or Intensive Care Unit (ICU).Thus, said patient group is in need of therapeutic intervention pursuantto the present disclosure, and thus in need for prevention or reductionof organ dysfunction or organ failure.

In another specific aspect, the patient group pursuant to the presentdisclosure is one having as a lower threshold at least three SOFAscores, being at 1 each for three of the clinical criteria respiration,and/or liver, and/or coagulation, and/or cardiovascular, and/or CNS,and/or renal on day of admission to hospital or Intensive Care Unit(ICU). Thus, said patient group is in need of therapeutic interventionpursuant to the present disclosure, and thus in need for prevention orreduction of organ dysfunction or organ failure.

In another specific aspect, the patient group pursuant to the presentdisclosure is one having as a lower threshold at least four SOFA scores,being at 1 each for four of the clinical criteria respiration, and/orliver, and/or coagulation, and/or cardiovascular, and/or CNS, and/orrenal on the day of admission to hospital or Intensive Care Unit (ICU).Thus, said patient group is in need of therapeutic intervention pursuantto the present disclosure, and thus in need for prevention or reductionof organ dysfunction or organ failure.

In another specific embodiment, the patient group in need of preventionor reduction of renal organ dysfunction or renal organ failure pursuantto the present disclosure is having a renal SOFA score of at least 1, orof 2, or of 3, or of 4.

In another specific embodiment, the patient group in need of preventionor reduction of liver organ dysfunction or liver organ failure pursuantto the present disclosure is having a liver SOFA score of at least 1, orof 2, or of 3, or of 4.

In another specific embodiment, the patient group in need of preventionor reduction of heart organ dysfunction or heart organ failure pursuantto the present disclosure is having a cardiovascular SOFA score of atleast 1, or of 2, or of 3, or of 4.

In another specific embodiment the patient group in need of preventionor reduction of lung organ dysfunction or lung organ failure pursuant tothe present disclosure is having a respiratory SOFA score of at least 1,or of 2, or of 3, or of 4.

Independent of the initial score, generally an increase in SOFA scoreduring the first 48 hours in the ICU or in the hospital predicts amortality rate of at least 50%.

Thus, in another specific embodiment the patient group in need oftherapeutic intervention for organ dysfunction/failure in accordancewith present disclosure is characterized by having at least one SOFAscore increased within the initial 48 hours after admission to hospitalor ICU.

In some aspects, the organ, organs or organ systems which is/are subjectto failure comprise at least one member of the following:cardiovascular, respiratory, renal, haematological, neurological,gastrointestinal organs, hepatic organs, heart, liver, lungs,intestines, colon, kidneys, spleen, and brain.

In some embodiments, the OCS is to be used in combination with fluidsadministered intravenously, wherein said combination is for use intherapy of a subject having a chronic or acute disease or acutecondition of a patient for protecting the organs of said patient. Thefluids to be administered intravenously are, of course, administeredsystemically.

In one embodiment, the subject having a chronic or acute disease orcondition being in need for protecting its organs is characterized bythe need of the subject to receive intravenous fluids.

The at least one OCS of the present disclosure may be applied for sakeof prevention or reduction of organ dysfunction and organ failure, andthus the at least one OCS is not necessarily intended for any methods ofprimary treatment or first line treatment to the chronic or acutedisease or acute condition itself, which therefore can be termed asunderlying disease(s). This means the present disclosure does notnecessarily provide for a therapy of healing/curing e.g. infections,cancer, or tumors located in the respective organ, but for resuscitatingthe respective organ towards physiologic function. Accordingly, thetherapy for a chronic or acute disease or acute condition of a patientwithin the scope of the present disclosure includes any kind of organinsufficiency, or poor organ function as an acute event.

Prevention and/or Treatment of Kidney Dysfunction and/or Failure

Kidney disease may be acute or chronic, or even acute-on-chronic renalfailure as discussed below.

Acute kidney injury (AKI, previously called acute renal failure (ARF))refers to an abrupt loss of kidney function that develops e.g. withinabout 7 days. AKI generally occurs because of damage to the kidneytissue caused by decreased renal blood flow (renal ischemia) from anycause e.g. low blood pressure, exposure to substances harmful to thekidney, an inflammatory process in the kidney, or an obstruction of theurinary tract which impedes the flow of urine. Causes of acute kidneyinjury include accidents, injuries, or complications from surgeries inwhich the kidneys are deprived of normal blood flow for extended periodsof time. Heart-bypass surgery is an example of one such procedure. Drugoverdoses, either accidental or from chemical overloads of drugs such asantibiotics or chemotherapy, may also cause the onset of acute kidneyinjury. AKI is diagnosed on the basis of characteristic laboratoryfindings, such as elevated blood urea nitrogen (BUN) and creatinine, orinability of the kidneys to produce sufficient amounts of urine (e.g.less than 400 mL per day in adults, less than 0.5 mL/kg/h in children orless than 1 mL/kg/h in infants). Thus, the present methods may includemeasuring or detecting one or more of these parameters in a subject and,if one or more or the measured parameters is positive and thusindicative of the presence of kidney malfunction developing within about7 days, then diagnosing acute kidney injury and administering at leastone OCS to the subject, as described herein.

Chronic kidney disease (CKD) usually develops slowly and, initially,patients may show few symptoms. CKD can be the long term consequence ofirreversible acute disease or part of a disease progression. CKD hasnumerous causes, including diabetes mellitus, long-term, uncontrolledhypertension, polycystic kidney disease, infectious diseases such ashantavirus, and certain genetic predisposition e.g. APOL1 gene variants.The present methods include administering at least one OCS to a subjecthaving CKD.

In some cases, the clinical criteria denoting the patient group(s) forkidney dysfunction/failure are as follows:

-   -   Patients at risk for kidney dysfunction/failure: GFR        decrease >25%, serum creatinine increased 1.5 times or urine        production of <0.5 ml/kg/hr for 6 hours    -   Patients with present kidney injury: GFR decrease >50%, doubling        of creatinine or urine production<0.5 ml/kg/hr for 12 hours    -   Patients with kidney failure: GFR decrease >75%, tripling of        creatinine or creatinine>355 μmol/l (with a rise of >44) (>4        mg/dl) or urine output below 0.3 ml/kg/hr for 24 hours    -   Patients with loss of kidney function: persistent acute kidney        injury (AKI) or complete loss of kidney function for more than 4        weeks    -   End-stage renal disease: complete loss of kidney function for        more than 3 months.

The overuse of drugs such as aspirin, ibuprofen, and acetaminophen(paracetamol) can also cause chronic kidney disease. This type of damagecan be avoided by administering these agents in combination with atleast one OCS, either via administration of the OCS in coordination withadministration of the agent (e.g. before or after or at the same timebut in a separate preparation); or, alternatively, by administeringcompositions comprising 1) liver-toxic drug such as aspirin, ibuprofen,and/or acetaminophen; and 2) at least one OCS. Accordingly, compositionscomprising aspirin plus one or more OCS are provided, as arecompositions comprising ibuprofen plus one or more OCS and compositionscomprising acetaminophen plus one or more OCS.

In compositions comprising aspirin plus one or more OCS, the aspirin isgenerally present in an approximate range of 80 mg to 1000 mg per unitdose (e.g. a single oral dosage form such as a pill, tablet, liquidetc.), i.e. about 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 mg. In compositionscomprising ibuprofen plus one or more OCS, the ibuprofen is present in arange of approximately 50 mg to 500 mg, usually approximately 100 mg to350 mg, and most usually approximately 125 mg to 250 mg per unit dose(e.g. in a single oral dosage form such as a pill, tablet, liquid,etc.). Exemplary doses of ibuprofen include 50, 60, 70, 80, 90, 100,150, 200, 250, 300, 350, 400, 450 and 500 mg. The dosage ofacetaminophen ranges from about 50 to about 4000 mg per dose, e.g. about50, 75, 100, 125, 150, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425,450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775,800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400,1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600,2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800,3900 or about 4000 mg/dose.

Such compositions may be prepared in solid forms such as tablets, pills,powders, suppositories, various slow- or extended-release formulations,and the like, or as liquid solutions, suspensions, emulsions, etc. orliquids suitable for injection and/or intravenous administration. Solidforms suitable for solution in, or suspension in, liquids prior toadministration may also be prepared. The active ingredients may be mixedwith excipients which are pharmaceutically acceptable and compatiblewith the active ingredients. Suitable excipients include, for example,water, saline, dextrose, glycerol, ethanol and the like, or combinationsthereof. In addition, the composition may contain minor amounts ofauxiliary substances such as wetting or emulsifying agents, pH bufferingagents, and the like. Oral dosage forms may include various thickeners,flavorings, diluents, emulsifiers, dispersing aids, binders, coatingsand the like. The compositions of the present disclosure may contain anysuch additional ingredients so as to provide the composition in a formsuitable for the intended route of administration. The final amount ofaspirin, ibuprofen and/or acetaminophen in a formulation may vary but ingeneral will be from about 1-99%. The final amount of OCS in aformulation may also vary but in general will be from about 1-99%, withparticular recommended doses being those described above. Still othersuitable formulations for use in the present disclosure can be found,for example in Remington's Pharmaceutical Sciences, Philadelphia, Pa.,19th ed. (1995); and Akers, Michael J. Sterile Drug Products:Formulation, Packaging, Manufacturing and Quality; publisher InformaHealthcare (2010).

Acetaminophen formulations that may be used in the compositions of thepresent disclosure (which also include at least one OCS such as 25HC3S)are described, for example, in U.S. Pat. Nos. 6,936,601; 6,926,907;6,924,273; 6,916,788; 6,855,310; 6,852,336; 6,841,544; 6,833,362;6,828,328; 6,787,164; 6,740,333; 6,702,850; 6,696,066; 6,686,390;6,642,243; 6,627,234; 6,622,856; 6,613,346; 6,602,518; 6,593,331;6,586,023; 6,569,439; 6,566,401; 6,566,361; 6,544,548; 6,528,097;6,524,623; 6,511,982; 6,509,380; 6,492,334; 6,485,747; 6,482,831;6,479,551; 6,475,526; 6,475,494; 6,458,809; 6,444,665; 6,440,983;6,429,223; 6,413,512; 6,406,716; 6,391,886; 6,391,337; 6,391,294;6,384,054; 6,383,527; 6,383,515; 6,375,957; 6,369,084; 6,369,082;6,355,666; 6,350,467; 6,335,034; 6,309,669; 6,306,842; 6,303,632;6,284,274; 6,277,384; 6,254,891; 6,245,802; 6,245,357; 6,242,493;6,225,295; 6,221,377; 6,217,911; 6,217,907; 6,214,386; 6,187,338;6,162,647; 6,159,500; 6,139,861; 6,127,352; 6,126,967; 6,077,533;6,077,530; 6,057,347; 6,054,451; 6,048,540; 6,028,222; 6,007,841;5,998,434; 5,972,916; 5,968,551; 5,965,167; 5,965,166; 5,945,416;5,942,530; 5,919,826; 5,914,129; 5,897,880; 5,891,801; 5,891,477;5,872,145; 5,863,922; 5,840,731; 5,837,729; 5,827,852; 5,776,462;5,773,031; 5,739,139; 5,733,578; 5,724,957; 5,654,334; 5,639,475;5,612,061; 5,603,959; 5,538,959; 5,474,757; 5,468,482; 5,466,865;5,458,879; 5,417,980; 5,409,944; 5,409,709; 5,336,691; 5,322,689;5,296,241; 5,273,759; 5,260,340; 5,238,686; 5,204,118; 5,154,926;5,100,675; 5,036,097; 5,023,085; 5,011,688; 4,971,960; 4,971,785;4,829,064; 4,822,781; 4,812,446; 4,794,112; 4,730,007; 4,703,045;4,478,822; 4,476,115; 4,466,960; 4,460,368; 4,401,665; 4,314,989;4,307,073; 4,260,629; 4,242,353; 4,237,140; 4,234,601; 4,233,317;4,233,316; 4,233,315; 4,233,314; 4,233,313; 4,207,340; 4,132,788 and4,049,803, and in pending US patent application 2012/0172324, thedisclosures of which are incorporated by reference in their entireties.

Contrast and enhancing dyes used for various types of imaging,especially iodine containing dyes, are also known to cause kidneydamage, especially in susceptible populations such as the elderly,diabetics, those who already have some form of kidney impairment, etc.Contrast-induced nephropathy is defined as either a greater than 25%increase of serum creatinine or an absolute increase in serum creatinineof 0.5 mg/dL in the wake of administration of a dye e.g. for X-rays orcomputed tomography (CT) scans. Iodine containing dyes include but arenot limited to iohexol, iodixanol and ioversol, as well as other ioniciodine dyes such as Diatrizoate (Hypaque 50), Metrizoate (Isopaque 370),and Ioxaglate (Hexabrix); and non-ionic contrast media such as Iopamidol(Isovue 370), Iohexol (Omnipaque 350), Ioxilan (Oxilan 350), Iopromide(Ultravist 370), and Iodixanol (Visipaque 320). The OCS described hereincan prevent or lessen the impact of such dyes when administered, forexample, before administration of the dye, and/or concomitantly with thedye and/or after dye administration to maintain kidney values at anormal level in spite of exposure to the dye, or to facilitate or speedthe return of those values to safe, normal ranges after dyeadministration.

Prevention and/or Treatment of Liver Dysfunction and/or Failure

An exemplary aspect of the present disclosure involves the treatment ofacute liver failure, especially acute liver failure caused by necrosis.Acute liver failure involves the rapid development of hepatocellulardysfunction, specifically coagulopathy and mental status changes(encephalopathy) in a patient without known prior liver disease. Thismalady embraces a number of conditions whose common thread is severeinjury of hepatocytes and/or massive necrosis e.g. loss of function of80-90% of liver cells. Loss of hepatocyte function sets in motion amultiorgan response characterized by the rapid appearance of severecomplications soon after the first signs of liver disease (such asjaundice). Complications include hepatic encephalopathy and impairedprotein synthesis, e.g. as measured by the levels of serum albumin andthe prothrombin time in the blood. Up to now, treatment options foracute liver failure have been limited and death often occurs suddenly,even after the liver has begun to recover from the original damage.

The diagnosis of acute liver failure (i.e. the identification of subjectexperiencing acute liver failure and who could benefit from the practiceof the present methods) is generally based on physical exam, laboratoryfindings, patient history, and past medical history to establish, forexample, mental status changes, coagulopathy, rapidity of onset, andabsence of known prior liver disease. The exact definition of “rapid”depends on the particular convention that is used. Differentsub-divisions exist which are based on the time from onset of firsthepatic symptoms to onset of encephalopathy. One scheme defines “acutehepatic failure” as the development of encephalopathy within 26 weeks ofthe onset of any hepatic symptoms. This is sub-divided into “fulminanthepatic failure”, which requires onset of encephalopathy within 8 weeks,and “subfulminant”, which describes onset of encephalopathy after 8weeks but before 26 weeks. Another scheme defines “hyperacute” liverfailure as onset within 7 days, “acute” liver failure as onset between 7and 28 days, and “subacute” liver failure as onset between 28 days and24 weeks. Subjects identified as experiencing acute liver failure by anyof these criteria may be treated by the methods described herein.

In some cases, the patient group for liver dysfunction/failure ischaracterized by a lower threshold of Bilirubin of >1.2 mg/dL,preferably >1.9 mg/dL, more preferably >5.9 mg/dL. Acute liver failurehas many potential causes and subjects identified as experiencing acuteliver failure for any reason can be treated by the methods describedherein. Possible causes include:

Acetaminophen (ATMP).

Taking too much acetaminophen (paracetamol, Tylenol®, others) is themost common cause of acute liver failure in the United States. Acuteliver failure can occur if a single very large dose of ATMP is taken allat once, or it can occur if higher-than-recommended doses are takenevery day for several days. People with chronic liver disease areespecially vulnerable, as are the elderly, the very young, etc. In suchsubjects, an ATMP “overdose” may be a dose that would be a safe ornormal dose for a person that does not have chronic liver disease or isnot elderly or very young. This aspect of the disclosure is discussed indetail below.

Prescription Medications.

Some prescription medications, including antibiotics, nonsteroidalanti-inflammatory drugs and anticonvulsants, can cause acute liverfailure.

Herbal Supplements.

Herbal drugs and supplements, including kava, ephedra, skullcap andpennyroyal, have been linked to acute liver failure.

Hepatitis and Other Viruses.

Hepatitis A, hepatitis B and hepatitis E can cause acute liver failure.Other viruses that can cause acute liver failure include Epstein-Barrvirus, cytomegalovirus and herpes simplex virus.

Toxins.

Toxins that can cause acute liver failure include the poisonous wildmushroom Amanita phalloides, which is sometimes mistaken for ediblespecies.

Autoimmune Disease.

Liver failure can be caused by autoimmune hepatitis, a disease in whichthe immune system attacks liver cells, causing inflammation and injury.

Diseases of the Veins in the Liver.

Vascular diseases, such as Budd-Chiari syndrome, can cause blockages toform in the veins of the liver and lead to acute liver failure.

Metabolic Disease.

Rare metabolic diseases, such as Wilson's disease and acute fatty liverof pregnancy, can cause acute liver failure.

Cancer.

Cancer that begins in the liver or cancer that spreads to the liver fromother locations in the body can cause acute liver failure.

Other.

Other causes include idiosyncratic reactions to medication (e.g.tetracycline, troglitazone), excessive alcohol intake (severe alcoholichepatitis), Reye syndrome (acute liver failure in a child with a viralinfection e.g. chickenpox in which aspirin may play a role; and others.Many cases of acute liver failure have no apparent cause.

Acute liver failure from any cause may be prevented and/or treated bythe methods and compositions of the present disclosure. The compositionsmay include at least one medicament or herbal supplement that ispotentially harmful to the liver plus at least one OCS such as 25HC3S.

In addition, various symptoms of liver toxicity may be prevented and/ortreated by the methods and compositions of the present disclosure priorto the development of full-blown ALF. Exemplary symptoms include but arenot limited to: cerebral edema and encephalopathy (which may lead tohepatic encephalopathy, coma, brain herniation, etc.); coagulopathy(e.g. prolongation in prothrombin time, platelet dysfunction,thrombocytopenia, intracerebral bleeding, etc.); renal failure (e.g. dueto original insult such as ATMP overdose resulting in acute tubularnecrosis, or from hyperdynamic circulation leading to hepatorenalsyndrome or functional renal failure); inflammation and infection (e.g.systemic inflammatory syndrome, which can lead to sepsis and multiorganfailure irrespective of the presence or absence of infection; variousmetabolic derangements such as hyponatremia, hypoglycemia, hypokalemia,hypophosphatemia, metabolic alkalosis, and lactic acidosis (occurringpredominantly in acetaminophen overdose); hemodynamic andcardio-respiratory compromise (e.g. hypotension, decrease in tissueoxygen uptake, tissue hypoxia and lactic acidosis); pulmonarycomplications (e.g. acute respiratory distress syndrome (ARDS), with orwithout sepsis, pulmonary haemorrhage, pleural effusions, atelectasis,and intrapulmonary shunts, etc.); late pregnancy complications, forwhich early clinical manifestations of ALF include hypodynamia, decreasein appetite, dark amber urine, deep jaundice, nausea, vomiting, andabdominal distention, etc. Subjects exhibiting one or more of thesesymptoms or conditions may benefit from the administration of at leastone OCS.

Acute Liver Failure Due to ATMP Toxicity

In some aspects, the present disclosure provides methods andcompositions for preventing and/or treating ATMP associated toxicity andsymptoms associated with or characteristic thereof, especially liverinjury or ALF as discussed above. ATMP toxicity is one of the mostcommon causes of poisoning worldwide and in the United States and theUnited Kingdom it is the most common cause of acute liver failure. Manyindividuals with ATMP toxicity may have no symptoms at all in the first24 hours following overdose. Others may initially have nonspecificcomplaints such as vague abdominal pain and nausea. With progressivedisease, signs of liver failure usually develop; these include low bloodsugar, low blood pH, easy bleeding, and hepatic encephalopathy. Damageto the liver, or hepatotoxicity, results not from ATMP itself, but fromone of its metabolites, N-acetyl-p-benzoquinoneimine (NAPQI), also knownas N-acetylimidoquinone. NAPQI depletes the liver's natural antioxidantglutathione and directly damages cells in the liver, leading to liverfailure. Risk factors for ATMP toxicity include excessive chronicalcohol intake, fasting or anorexia nervosa, and the use of certaindrugs such as isoniazid.

Data presented herein show that administration of 25HC3S dramaticallyreduces mortality in subjects suffering from acetaminophen(ATMP)-induced acute liver failure. Methods to prevent or treat ALF in asubject in need thereof, especially liver dysfunction and/or acute liverfailure associated with ATMP toxicity, are described in this disclosure.The methods may include administering at least one OCS (e.g. 25HC3S)prior to administration of ATMP, and/or concomitantly withadministration of ATMP, and/or after administration of ATMP, to preventand/or treat ATMP toxicity.

The disclosure also provides new compositions of matter which compriseacetaminophen co-formulated with at least one OCS, described above under“kidney dysfunction and failure”. The at least one OCS is present in thecomposition in an amount sufficient to prevent (or at least lessen)toxicity of the acetaminophen in a subject to whom the composition isadministered. The compositions include at least one substantiallypurified OCS, acetaminophen and one or more pharmacologically suitablecarriers.

Prevention and/or Treatment of Pancreas Dysfunction and Failure

The pancreas is a glandular organ that functions in the digestive systemand endocrine system of vertebrates. It produces several importanthormones, including insulin, glucagon, somatostatin, and pancreaticpolypeptide, and also secretes pancreatic juice containing digestiveenzymes that assist digestion and absorption of nutrients in the smallintestine. Inflammation of the pancreas (pancreatitis) has severalcauses and typically requires immediate treatment. It may be acute,beginning suddenly and lasting a few days, or chronic, occurring overmany years. Eighty percent of cases of pancreatitis are caused byalcohol or gallstones, with gallstones being the single most commonetiology of acute pancreatitis and alcohol being the single most commonetiology of chronic pancreatitis. Severe pancreatitis is associated withorgan failure, necrosis, infected necrosis, pseudocyst and abscess,having mortality rates around 2-9%, and higher where necrosis hasoccurred. Severe pancreatitis is diagnosed if at least three of thefollowing are true: patient age is greater than 55 years; blood PO2oxygen is less than 60 mm Hg or 7.9 kP; white blood cells>15,000 WBCsper microliter (mcL); calcium<2 mmol/L; urea>16 mmol/L; lactatedehydrogenase (LDH)>600 iu/L; aspartate transaminase (AST)>200 iu/L;albumin<32 g/L; and glucose>10 mmol/L.

An aspect of the present disclosure is the treatment of pancreaticdysfunction and/or failure by administering at least one OCS to apatient in need thereof. Suitable patients or patient populations areidentified, by a skilled medical practitioner, as exhibiting at leastone of the symptoms or criteria listed above.

Prevention and/or Treatment of Heart Dysfunction and Failure

Heart failure (HF), often used to mean chronic heart failure (CHF),occurs when the heart is unable to pump sufficiently to maintain bloodflow to meet the needs of the body. The terms congestive heart failure(CHF) or congestive cardiac failure (CCF) are often used interchangeablywith chronic heart failure. Symptoms commonly include shortness ofbreath (especially with exercise, when lying down, and at night whilesleeping), excessive tiredness, and leg swelling. Common causes of heartfailure include coronary artery disease including a previous myocardialinfarction (heart attack), high blood pressure, atrial fibrillation,valvular heart disease, and cardiomyopathy. Heart failure is distinctfrom myocardial infarction, in which part of the heart muscle dies, andcardiac arrest, in which blood flow stops altogether.

Heart failure is typically diagnosed based on the history of thesymptoms and a physical examination with confirmation byechocardiography, blood tests, and/or chest radiography.Echocardiography uses ultrasound to determine the stroke volume (SV, theamount of blood in the heart that exits the ventricles with each beat),the end-diastolic volume (EDV, the total amount of blood at the end ofdiastole), and the SV in proportion to the EDV, a value known as theejection fraction (EF). Abnormalities in one or more of these mayindicate or confirm heart dysfunction and/or failure. Anelectrocardiogram (ECG/EKG) is used to identify arrhythmias, ischemicheart disease, right and left ventricular hypertrophy, and presence ofconduction delay or abnormalities (e.g. left bundle branch block).Abnormalities in one or more of these may also indicate or confirm heartdysfunction and/or failure. Blood tests routinely performed to diagnoseor confirm heart dysfunction/failure include electrolytes (sodium,potassium), measures of renal function, liver function tests, thyroidfunction tests, a complete blood count, and often C-reactive protein ifinfection is suspected. Abnormalities in one or more of these may alsoindicate or confirm the presence of heart dysfunction and/or failure. Anelevated B-type natriuretic peptide (BNP) is a specific test indicativeof heart failure. If myocardial infarction is suspected, various cardiacmarkers may be tested, including but not limited to troponin creatinekinase (CK)-MB (an isoform of creatine kinase); lactate dehydrogenase;aspartate transaminase (AST) (also referred to as aspartateaminotransferase); myoglobin; ischemia-modified albumin (IMA); pro-brainnatriuretic peptide; glycogen phosphorylase isoenzyme BB, etc. Abnormallevels of one or more of these (usually abnormally high levels) areconsidered as identifying a subject in need of treatment for cardiacdysfunction or failure.

A subject who is confirmed to have or suspected of having cardiacdysfunction or failure is treated by administration of a therapeuticallyeffective amount of at least one OCS as described herein (e.g. 25HC3S),the amount being sufficient to prevent symptoms of heart dysfunction orfailure, or to ameliorate symptoms of heart dysfunction or failure, e.g.to at least partially restore heart function to normal or near normal,and/or to prevent further deterioration of heart function and health ofthe patient.

Prevention and/or Treatment of Brain Dysfunction and Failure

Brain dysfunction and/or failure (i.e. organic brain syndrome “OBS”) isa general term that describes decreased mental function due to a medicaldisease other than a psychiatric illness. Causes include but are notlimited to brain injury caused by trauma; bleeding into the brain(intracerebral hemorrhage); bleeding into the space around the brain(subarachnoid hemorrhage); blood clot inside the skull causing pressureon brain (subdural hematoma); concussion; various breathing conditionssuch as low oxygen in the body (hypoxia) and high carbon dioxide levelsin the body (hypercapnia); various cardiovascular disorders, e.g.dementia due to many strokes or multi-infarct dementia, heart infections(endocarditis, myocarditis), stroke (e.g. spontaneous stroke) andtransient ischemic attack (TIA) or so-called “ministrokes”; or due tovarious degenerative disorders such as Alzheimer disease,Creutzfeldt-Jacob disease, diffuse Lewy Body disease, Huntingtondisease, multiple sclerosis, normal pressure hydrocephalus, Parkinsondisease and Pick disease; dementia due to metabolic causes such askidney, liver, or thyroid disease and/orvitamin deficiency (B1, B12, orfolate); as well as drug and alcohol-related conditions e.g. alcoholwithdrawal state, intoxication from drug or alcohol use,Wernicke-Korsakoff syndrome (a long-term effect of excessive alcoholconsumption or malnutrition), and withdrawal from drugs (especiallysedative-hypnotics and corticosteroids); and sudden onset (acute) orlong-term (chronic) infections e.g. septicemia, encephalitis,meningitis, prion infections, and late-stage syphilis; as well ascomplications of cancer or cancer treatment. Symptoms of OBS includeagitation, confusion; long-term loss of brain function (dementia), andsevere, short-term loss of brain function (delirium), as well as impactson the autonomic nervous system which controls e.g. breathing. Diagnosisor confirmation of the presence of OBS is determined by detecting ormeasuring various methodology such as blood tests, electroencephalogram(EEG), head CT scan, head MRI and/or lumbar puncture [for which normalvalues typically range as follows: pressure: 70-180 mm Hg; cerebralspinal fluid (CSF) appearance: clear, colorless; CSF total protein:15-60 mg/100 mL; gamma globulin: 3-12% of the total protein; CSFglucose: 50-80 mg/100 mL (or greater than ⅔ of blood sugar level); CSFcell count: 0-5 white blood cells (all mononuclear), and no red bloodcells; and CSF chloride: 110-125 mEq/L).

If one or more of these tests or analyses or indicia are abnormal, thesubject is generally considered as susceptible to or already sufferingfrom OBS. A subject who is confirmed to have or suspected of having OBS(either early stage or advanced) is treated by administration of atherapeutically effective amount of at least one OCS as described herein(e.g. 25HC3S), the amount being sufficient to prevent symptoms of OBS,or to ameliorate symptoms of OBS, e.g. to at least partially restorebrain function to normal or near normal, and/or to prevent furtherdeterioration of brain function and health of the patient.

Heart failure may also occur as a side effect and/or in the aftermath ofchemotherapy, e.g. chemotherapy received as treatment for cancer such asbreast cancer. The administration of at least one OCS as describedherein to a patient receiving or who has already received chemotherapymay prevent unwanted damage to heart (and other organs, organ systems,tissues and cells) during or after cancer chemotherapy. In other words,the at least one OCS is used as a protective agent for deleteriouseffects of chemotherapy.

Organ Dysfunction and/or Failure Due to Trauma

In some aspects, the organ dysfunction/failure is due to trauma.Examples of trauma injuries include but are not limited to: woundsresulting from vehicular accidents; gunshot wounds (both accidentalduring hunting associated activities, and intentionally inflicted suchas those associated with criminal activity or war); blunt trauma orblunt injury e.g. non-penetrating blunt force trauma such as physicaltrauma to a body part e.g. by impact, injury or physical attack; etc.Examples of blunt trauma include but are not limited to: concussion,e.g. concussion suffered by athletes or by persons involved inaccidents, falls, etc., and blunt trauma suffered as the result of anencounter with a projectile such as a falling object, and others.

Individuals who are susceptible to such blunt trauma (e.g. athletes, theelderly) may benefit from prophylactic administration of one or moreOCS, and if blunt trauma such as a concussion is diagnosed in a subject,the subject will benefit by administration as soon as possible after theinjury is suspected or confirmed.

Prevention and/or Treatment of Conditions Caused by Ischemia

Ischemia refers to an insufficient supply of blood to a tissue or organ,causing a shortage of oxygen and glucose needed for cellular metabolismand to keep tissue alive. Hypoxia (also known as hypoxiation oranoxemia) is caused by ischemia and refers to the condition in which thebody or a region of the body is deprived of adequate oxygen supply.Ischemia results in tissue damage in a process known as the ischemiccascade. Damage is largely the result of the build-up of metabolic wasteproducts, the inability to maintain cell membranes, mitochondrialdamage, and eventual leakage of autolyzing proteolytic enzymes into thecell and surrounding tissues. Ensuing inflammation also damages cellsand tissues. Without immediate intervention, ischemia may progressquickly to tissue necrosis, and ultimately to, for example, organdysfunction or failure.

In addition, restoration of blood supply to ischemic tissues can causeadditional damage known as reperfusion injury. Reperfusion injury can bemore damaging than the initial ischemia. Reintroduction of blood flowbrings oxygen back to the tissues, causing a greater production of freeradicals and reactive oxygen species that damage cells. It also bringsmore calcium ions to the tissues, which may cause calcium overloadingand can result in potentially fatal cardiac arrhythmias, and which mayaccelerate cellular self-destruction. The restored blood flow may alsoexaggerate the inflammation response of damaged tissues, causing whiteblood cells to destroy damaged but still viable cells.

The present disclosure provides methods of preventing and/or treatingthe untoward effects or outcomes of ischemia, includingischemia/reperfusion injury, in a subject in need thereof. The methodsmay comprise administering a therapeutically effective amount of one ormore OCS sufficient to prevent or treat symptoms of ischemia and/orischemia/reperfusion. The methods may also include identifying ordiagnosing a subject who will experience, or is experiencing or who hasexperienced ischemia and/or ischemia/reperfusion. The ischemia and/orischemia/reperfusion may be due to a disease process (e.g.artherosclerosis, a blood clot, etc.), or due to an accident (e.g.severing of an artery or other blood conduit), or may be intentional(planned), e.g. as occurs during some heart or other surgeries in orderto temporarily stop blood flow to a defined or circumscribed region ofthe body.

Types of ischemia that are relevant to the methods described hereininclude but are not limited to:

Cardiac ischemia, e.g., myocardial ischemia, occurring when the heartmuscle, or myocardium, receives insufficient blood flow. This mostfrequently results from atherosclerosis, which is the long-termaccumulation of cholesterol-rich plaques in coronary arteries.Bowel ischemia: Both large and small bowel can be affected by ischemicinjury. Ischemic injury of the large intestine may result in aninflammatory process known as ischemic colitis and also as a result ofsurgery and adhesion development. Ischemia of the small bowel is calledmesenteric ischemia.Brain ischemia is insufficient blood flow to the brain, and can be acute(i.e., rapid) or chronic (i.e., long-lasting). Acute ischemic stroke isa neurologic emergency that may be reversible if treated rapidly.Chronic ischemia of the brain may result in a form of dementia calledvascular dementia. A brief episode of ischemia affecting the brain iscalled a transient ischemic attack (TIA), often erroneously referred toas a “mini-stroke”.Limb ischemia: Lack of blood flow to a limb results in acute limbischemia.Cutaneous ischemia refers to reduced blood flow to the skin layers,which may result in mottling or uneven, patchy discoloration of theskin, and may lead to the development of cyanosis, or other conditionssuch as pressures sores (e.g. decubitus ulcers, bedsores, etc.).Reversible ischemia refers to a condition which results in a lack ofblood flow to a particular organ which can be reversed through use ofmedications or surgery. It most often refers to hindered blood flow tothe heart muscle, but it can refer to an obstruction blocking any organin the body, including the brain. Whether or not a case of ischemia canbe reversed will depend on the underlying cause. Plaque buildup in thearteries, weakened arteries, low blood pressure, blood clots, andunusual heart rhythms can all be causes of reversible ischemia.Apical ischemia refers to lack of blood flow to the apex or bottom tipof the heart.Mesenteric ischemia refers to inflammation and injury of the smallintestine occurs due to inadequate blood supply. Causes of the reducedblood flow can include changes in the systemic circulation (e.g. lowblood pressure) or local factors such as constriction of blood vesselsor a blood clot.Ischemia of various organs, including but not limited to liver (hepaticischemia), kidney, intestines, etc.

Ischemia, ischemia/reperfusion may also be causally related toinflammation and organ dysfunction/failure. For example, cerebral(brain) ischemia is typically accompanied by a marked inflammatoryreaction that is initiated by ischemia-induced expression of cytokines,adhesion molecules, and other inflammatory mediators, includingprostanoids and nitric oxide. It is known that interventions aimed atattenuating such inflammation reduce the progression of brain damagethat occurs e.g. during the late stages of cerebral ischemia. Inaddition, the most frequent cause of intrarenal (kidney) failure (ARF)is transient or prolonged renal hypoperfusion (ischemia).

Other types of ischemia, the effects of which can be treated orprevented as described herein, include but are not limited to: ischemicstroke, small vessel ischemia, ischemia/reperfusion injuries, etc.

Diagnosis of ischemia is generally carried out by identifying one ormore symptoms of malfunction in the particular organ or organ system ortissue or cell that is affected. Thus, symptoms include those listedherein for dysfunction/failure of individual organs, plus documentationof ischemia per se, such as by noting the history of the patient (e.g.known occlusion, blockage or severance of an artery that otherwisesupplies blood to the organ or tissue, imaging which shows or isconsistent with such observations, etc.

If one or more suitable tests or analyses or indicia are abnormal, thesubject is generally considered as susceptible to or already sufferingfrom ischemia. A subject who is confirmed to have or suspected of havingischemia (or is known to be undergoing future planned ischemia, e.g.during a surgical procedure) may be treated by administration of atherapeutically effective amount of at least one OCS as described herein(e.g. 25HC3S), the amount being sufficient to prevent symptoms ofischemia and/or ischemia-reperfusion injury, or to ameliorate symptomsof ischemia and/or ischemia-reperfusion injury, e.g. to at leastpartially restore organ or tissue function to normal or near normal whenblood flow is reestablished, and/or to prevent further deterioration oforgan or tissue function and health of the patient.

Prevention and/or Treatment of Effects of Unwanted Cell Death

Active, regulated cell death is referred to as “programmed cell-death”or “PCD” and is a regulated process mediated by intracellular pathways.While PCD is generally beneficial to an organism, aberrations insignaling or the presence of overwhelming stresses on the cell may causeundesirable PCD to occur. The forms of PCD include apoptosis, theinitiation of controlled intracellular signaling in response to astress, which brings about cell suicide; and necroptosis, a form of PCDthat serves as a backup to apoptosis, e.g. when the apoptosis signalingis blocked by endogenous or exogenous factors such as viruses ormutations.

In contrast to PCD, necrosis refers to unregulated, passive cell deathwhich results in the harmful, premature death of cells in living tissue.Necrosis is typically caused by factors external to the cell or tissue,such as infection, toxins, trauma, ischemia, etc. Without being bound bytheory, it is believed that necrosis involves the loss of cell membraneintegrity and an uncontrolled release of products of cell death into theintracellular space, thereby initiating an inflammatory response in thesurrounding tissue which prevents nearby phagocytes from locating andeliminating the dead cells by phagocytosis. While surgical removal ofnecrotic tissue can halt the spread of necrosis, in some cases surgicalintervention is not possible or practical e.g. when internal tissues ororgans are involved. Thus, necrosis of internal organs often leads todangerous and often deadly organ dysfunction and/or failure.

The present disclosure provides methods of preventing and/or treatingthe effects of unwanted cell death in a subject in need thereof,especially unwanted apoptosis and necrosis associated with organdysfunction and/or organ failure. The cell death may result from or beassociated with unwanted PCD (e.g. unwanted or deleterious apoptosis,autophagy, or necroptosis) or with necrosis, which is unwanted bydefinition; and/or combinations of these. The methods compriseadministering a therapeutically effective amount of one or more OCS, theamount being sufficient to prevent unwanted cell death from occurring,or to treat the effects of unwanted cell death that has already occurredin a subject.

Unwanted or deleterious cell death via apoptosis occurs, for example, inthe aftermath of ischemia and in Alzheimer's disease. Unwanted apoptosisis extremely harmful, causing extensive tissue damage.

Types of necrosis that may be prevented and/or treated by the methodsdescribed herein include but are not limited to:

Aseptic necrosis is necrosis without infection, usually in the head ofthe femur after traumatic hip dislocation.

Acute tubular necrosis refers to acute renal failure with mild to severedamage or necrosis of tubule cells, usually secondary to eithernephrotoxicity, ischemia after major surgery, trauma (crush syndrome),severe hypovolemia, sepsis, or burns.

Avascular necrosis is the consequence of temporary or permanentcessation of blood flow to the bones. The absence of blood causes thebone tissue to die, resulting in fracture or collapse of the entirebone.

Balser's fatty necrosis is gangrenous pancreatitis with omental bursitisand disseminated patches of necrosis of fatty tissues.

Bridging necrosis is necrosis of the septa of confluent necrosisbridging adjacent central veins of hepatic lobules and portal triadscharacteristic of subacute hepatic necrosis.

Caseous or “cheesy” necrosis is necrosis in which the tissue is soft,dry, and cottage cheese-like, most often seen in tuberculosis andsyphilis; in contrast to moist necrosis in which the dead tissue is wetand soft.

Central necrosis is necrosis affecting the central portion of anaffected bone, cell or lobule of the liver.

Coagulation necrosis refers to necrosis of a portion of an organ ortissue, with formation of fibrous infarcts, the protoplasm of the cellsbecoming fixed and opaque by coagulation of the protein elements, thecellular outline persisting for a long time.

Colliquative or liquefaction necrosis is that in which the necroticmaterial becomes softened and liquefied.

Contraction band necrosis refers to a cardiac lesion characterized byhypercontracted myofibrils and contraction bands, and mitochondrialdamage caused by calcium influx into dying cells resulting in arrest ofthe cells in the contracted state.

Fat necrosis is that in which the neutral fats in adipose tissue arebroken down into fatty acids and glycerol, usually affecting thepancreas and peripancreatic fat in acute hemorrhagic pancreatitis.

Gangrenous necrosis is that is which ischemia combined with bacterialaction causes putrefaction to set in. “Gangrene” includes dry gangrene,wet gangrene, gas gangrene, internal gangrene and necrotizing fasciitis.

Gingival necrosis refers to the death and degeneration of the cells andother structural elements of the gingivae (e.g., necrotizing ulcerativegingivitis).

Interdental necrosis is a progressive disease that destroys the tissueof the papillae and creates interdental craters. Advanced interdentalnecrosis leads to a loss of periodontal attachment.

Ischemic necrosis refers to death and disintegration of a tissueresulting from interference with its blood supply, thus depriving thetissues of access to substances necessary for metabolic sustenance.

Macular degeneration: Macular degeneration (both wet and dry forms)occurs when the small central portion of the retina, known as themacula, deteriorates. Because the disease develops as a person ages, itis often referred to as age-related macular degeneration (AMD).Massive hepatic necrosis refers to massive, usually fatal, necrosis ofthe liver, a rare complication of viral hepatitis (fulminant hepatitis)that may also result from exposure to hepatotoxins or from drughypersensitivity.Phosphorus necrosis is necrosis of the jaw bone due to exposure tophosphorus.Postpartum pituitary necrosis refers to necrosis of the pituitary duringthe postpartum period, often associated with shock and excessive uterinebleeding during delivery, and leading to variable patterns ofhypopituitarism.Radiation necrosis is the death of tissue caused by radiation.Selective myocardial cell necrosis refers to myofibrillar degeneration.Zenker's necrosis refers to hyaline degeneration and necrosis ofstriated muscle; also called Zenker's degeneration.

Such unwanted or pathological cell death may be prevented or treated bycontacting affected cells with one or more OCSs in an amount sufficientto prevent or treat death of the cells, and or to prevent the spread ofcell death signaling to adjacent cells. Candidate cells for treatment,or organs containing candidate cells for treatment, are identified byany or several known techniques, e.g. by observation of overt effects ofcell death (tissue breakdown, liquification, odor, etc.), detectingrelease of lactate dehydrogenase (LDH), by various scans such astomography or nuclear magnetic resonance, by detecting the presence ofcausative bacteria (e.g. using PCR), using antibodies, etc.

Prevention and/or Treatment of Symptoms Related to or Caused by Sepsis(Inflammatory Response Syndrome, or SIRS)

Sepsis is a potentially life-threatening whole-body inflammation causedby a serious infection which triggers an immune response. The infectionis typically caused by bacteria, but can also be due to fungi, viruses,or parasites in the blood, urinary tract, lungs, skin, or other tissues.Unfortunately, symptoms can continue even after the infection is gone.Severe sepsis is sepsis causing poor organ function or insufficientblood flow as evidenced e.g. by low blood pressure, high blood lactate,and/or low urine output. In fact, sepsis is considered to fall within acontinuum from infection to multiple organ dysfunction syndrome (MODS).Septic shock is low blood pressure due to sepsis that does not improveafter reasonable amounts of intravenous fluids are given.

Up to now, sepsis was typically treated with intravenous fluids andantibiotics, often in an intensive care unit. Various medications andother interventions may be used, e.g. mechanical ventilation, dialysis,and oxygen saturation may also be used. Outcomes depend on the severityof disease with the risk of death from sepsis being as high as 30%,severe sepsis as high as 50%, and septic shock as high as 80%.

Provided herein are methods of preventing or treating sepsis byadministering to a subject or patient in need thereof, a therapeuticallyeffective amount of at least one OCS. For instance, the presentdisclosure includes the treatment of mammalian endotoxemia andsepticemia and renal and mesenteric vasoconstriction that is induced bycatecholamines that are used to treat endotoxemia and septic shock. Theterm “endotoxemia” refers to the presence of microbial endotoxins in thebloodstream. Subjects inflicted with endotoxemia usually also havesepticemia. The present disclosure includes a method for treatingsepticemia/endotoxemia. The present disclosure also includes a methodfor treating acute renal failure caused by septicemia/endotoxemia.Further, the present disclosure includes a method for treating renalvasoconstriction caused by septicemia/endotoxemia. Still further, thepresent disclosure provides a method for attenuatingcatecholamine-induced renal and mesenteric vasoconstriction. Yetfurther, the present disclosure includes a method to prevent damage to apatient's intestines and kidney due to the effects of endotoxin and/orvasopressor agents.

Sepsis is associated with mitochondrial dysfunction, which leads toimpaired oxygen consumption and may lead to sepsis-induced multipleorgan failure. This holds especially true for raised tissue oxygentensions in septic patients, suggesting reduced ability of the organs touse oxygen. Because ATP production by mitochondrial oxidativephosphorylation accounts for more than 90% of total oxygen consumptionmitochondrial dysfunction may directly results in organ failure,possibly due to nitric oxide, which is known to inhibit mitochondrialrespiration in vitro and is produced in excess in sepsis. Therefore, ina specific embodiment of the present disclosure, the OCS is used inmethods of prevention for organ dysfunction and failure in SystemicInflammatory Response-Syndrome (SIRS), sepsis, severe sepsis, and septicshock patients.

The methods may include identifying a suitable patient in need of suchtreatment, e.g. by detecting or measuring at least one symptom ofsepsis, e.g. abnormal temperature (body temperature above 101 F (38.3 C,“fever”) or below 96.8 F (36 C), increased heart rate, increasedbreathing rate, probable or confirmed infection, and possibly confusion.Patients with severe sepsis exhibit at least one of the following signsand symptoms, which indicate an organ may be failing: significantlydecreased urine output, abrupt change in mental status, decrease inplatelet count, difficulty breathing, abnormal heart pumping function,and abdominal pain. A diagnosis of septic shock is generally based onobserving the signs and symptoms of severe sepsis plus measuringextremely low blood pressure that does not adequately respond to simplefluid replacement.

In some cases, a subject may be a candidate for prophylactic ortherapeutic treatment with OCS of sepsis is based on cough/sputum/chestpain; abdominal pain/distension/diarrhea; line infection; endocarditis;dysuria; headache with neck stiffness; cellulitis/wound/joint infection;and/or positive microbiology for any infection.

In other cases, a subject may be a candidate for prophylactic ortherapeutic treatment with OCS of severe sepsis based on a diagnosis ofsepsis and at least one clinical suspicion of any organ dysfunctionselected from: blood pressure systolic<90/mean; <65 mm HG; lactate>2mmol/L; Bilirubine>34 μmol/L; urine output<0.5 mL/kg/h for 2 h;creatinine>177 μmol/L; platelets<100×10⁹/L; and SpO₂>90% unless O₂given.

In some cases, a subject may be a candidate for prophylactic ortherapeutic treatment with OCS of septic shock if there is refractoryhypotension that does not respond to treatment and intravenous systemicfluid administration alone is insufficient to maintain a patient's bloodpressure from becoming hypotensive.

Patients with a diagnosis of (exhibiting signs of) early sepsis, severesepsis or septic shock are candidates for treatment with the OCSdescribed herein, e.g. by administration of a therapeutically effectiveamount of at least one OCS as described herein (e.g. 25HC3S). The amountadministered may be sufficient to prevent symptoms of sepsis fromdeveloping or continuing, or to at least lessen the impact of symptomsof sepsis.

Prevention of Post-Harvest Damage to Harvested Organs, Tissues and Cells

Provided herein are methods, compositions and systems (e.g. apparatuses)for preserving the viability and/or preventing (protecting against)damage to and deterioration of extracorporeal cells, tissues or organs.In some aspects, the length of time for which an organ or tissue isuseable for transplant may be extended, and/or organs that are otherwisenot suitable for transplant may be “rescued”, by contact with one ormore OCS. In some aspects, the cells, tissues or organs are harvestedfrom a donor and are ex vivo. The donor may be a living donor or acadaver, and may be of any species, although frequently the donor is amammal such as a human. In other aspects, the cells, tissues or organsare engineered, i.e. artificially generated by growth under controlledconditions in a laboratory. In some aspects, the organs are intended tobe implanted or grafted into a transplant recipient. The recipient maybe of any species, although frequently the recipient is a mammal such asa human. In some aspects, the cells, tissues and/or organs are notintended for transplant into a living donor per se but are intended foruse in experimental procedures.

Application of the OCSs generally occurs after the material that is tobe transplanted is removed from the body of a donor, especially if thedonor is a live donor; however, if removal is from a cadaver,application may occur at any time after death of the donor is confirmed.In such cases, the OCS may be provided to several cells, tissues and/ororgans at the same time within the cadaver, e.g. by artificiallycirculating or pumping a composition comprising the OCS through thecirculatory or other system of the cadaver, or in a directed mannerthrough one or more tissues or organs to be harvested until the materialto be transplanted is harvested. If the material is artificiallygenerated, application/contact may occur at any convenient stage ofdevelopment or use of the material.

The methods generally involve contacting a harvested cell, tissue ororgan with one or more OCS e.g. by applying the one or more OCS to thecell, tissue or organ, e.g. by bathing, flushing or submerging the cell,tissue or organ in or with a composition comprising the one or more OCS,and/or, in the case of tissue and organs, by perfusing the tissue ororgan by pumping or circulating the composition into and through thetissue or organ. Suitable OCS for use in the composition include but arenot limited to 5-cholesten-3,25-diol, 3-sulfate (25HC3S) and5-cholesten, 3b, 25-diol, disulfate (25HCDS); (5-cholestene, 3, 27-diol,3-sulfate); (5-cholestene, 3, 27-diol, 3, 27-disulfate); (5-cholestene,3,7-diol, 3-sulfate); (5-cholestene, 3,7-diol, 3,7-disulfate);(5-cholestene, 3, 24-diol, 3-sulfate); (5-cholestene, 3, 24-diol, 3,24-disulfate); and (5-cholestene, 3-ol, 24, 25-epoxy 3-sulfate).

In general, the one or more OCS are present in a suitable biologicallycompatible liquid carrier or solution (medium), and may be appropriatefor either cold (e.g. 0-4° C.) or warm (e.g. up to about 37° C.)transport and/or storage.

The transplant medium including the at least one OCS is typicallyaqueous. In addition to the at least one OCS, the aqueous medium mayinclude at least one of electrolytes, buffers, impermeants, colloids,ROS scavengers, and substrates. Examples of electrolytes include, butare not limited to, calcium, chloride, magnesium, phosphate, potassium,sodium, and sulphate. Examples of buffers include, but are not limitedto, citrate, histidine, K₂HPO₄, KH₂PO₄, Na₂HPO₄, NaHCO₃, and NaH₂PO₄.Examples of impermeants include, but are not limited to, glucose,histidine, lactobionate, mannitol, raffinose, and sucrose. Examplescolloids include, but are not limited to, hydroxyethyl starch (HES) andpolyethylene glycol (PEG). Examples of ROS scavengers include, but arenot limited to, allopurinol, glutathionine, mannitol, and tryptophan.Examples of substrates include, but are not limited to, adenosine,glutamate, and ketoglutarate.

In some cases, the aqueous medium includes at least one OCS and at leastone of lactobionate, raffinose, HES, steroids, and insulin. In othercases, the aqueous medium includes at least one OCS and at least one ofpotassium lactobionate, KH₂PO₄, MgSO₄, raffinose, adenosine,glutathionine, allopurinol, and HES. In some cases, the aqueous mediumincludes at least one OCS and at least one of a phosphate buffer andglucose. In certain cases, the aqueous medium includes at least one OCSand at least one of phosphate buffer and sucrose. In some cases, theaqueous medium includes at least one OCS and at least one of citratebuffer, mannitol, citrate, and magnesium. In some cases, the aqueousmedium includes at least one OCS and at least one of histidine buffer,mannitol, and histidine bionate. In certain cases, the aqueous mediumincludes at least one OCS and at least one of phosphate buffer,raffinose, and lactobionate. In some case, the aqueous medium includesat least one OCS and at least one phosphate buffer, mannitol, andlactobionate. In other cases, the aqueous medium includes at least oneOCS and at least one of trehalose, gluconate, and HES. In still othercases, the aqueous medium includes potassium lactobionate, KH₂PO₄,MgSO₄, raffinose, adenosine, glutathionine, allopurinol, and PEG. Insome cases, the aqueous medium includes a scavenger, such as at leastone of mannitol and glutathion. In some cases, the aqueous mediumincludes at least one of ketoglutarate, adenosine, and glutamate.

The aqueous medium typically has a pH ranging from about 7.1 to about7.4. The aqueous medium typically has an osmolality ranging from about300 to about 400, such as from 310 to about 390.

Some transplant media are oxygenated (e.g. using oxygen microbubbles,see U.S. Pat. No. 7,749,692). The media may include various biologicallycompatible preservatives such as cryopreservatives. The media maycomprise or be whole blood. Examples of suitable media include but arenot limited to: Steen Solution™; Perfadex®;Histidine-tryptophan-ketoglutarate solution or Custodiol HTK™; Viaspan™;TransMedics Solutions; and those described in U.S. Pat. Nos. 8,409,846;7,981,596; 7,977,383; 7,592,023; 7,537,885; 7,476,660; 6,365,338; and5,306,711; or in published US patents 2014/0234827; 2013/0102059;2011/0300237; 2010/0272789; and 2005/0164156, the complete contents ofeach of which are hereby incorporated by reference in their entireties.The one or more OCS are generally present in the medium at aconcentration of from about 1 to about 1000 mg/liter (e.g. about 0.05 mMto about 50 mM). In a further aspect, what is provided is an in vitrocomposition comprising 1) one or more of: a cell or a plurality ofcells; tissue or a plurality of tissues; and/or at least one organ ororgan system; 2) one or more OCS; and 3) a biologically compatiblemedium, such as one of the above-noted aqueous media. In yet a furtheraspect, what is provided is a medium or composition comprising one ormore OCS and a biologically compatible carrier, such as one of theabove-noted aqueous media.

The compositions may be incorporated into an organ preservation and/ortransport system, which includes a container for containing the cells,tissues or organs and the preservation media, and, optionally, amechanism for circulating or pumping the medium. The system may beportable. The compositions may be incorporated into existing systems(e.g. TransMedics' proprietary Organ Care System, XVIVO's Lung Perfusionsystem, etc., or may be included in a newly developed system.

Exemplary organs which are treated and transported in this mannerinclude but are not limited to kidney (single, en bloc and doublekidney), heart, heart and lung together, liver (including portionsthereof, right or left lobes, and lateral or other segments), lung(including single lungs, double lungs, and lung lobes), pancreas (whichmay include a spleen and splenic artery), stomach, etc. The organs,tissues and cells may be allografts, isografts or even xenografts (e.g.porcine heart valves used for transplant into humans). In some cases,the organ(s) which are treated and transported in this manner do notinclude liver. Exemplary tissues which are treated and transported inthis manner include but are not limited to bones, tendons, ligaments,skin, heart valves, blood vessels, corneas, nerve tissue, etc. Exemplarycells which are treated and transported in this manner include but arenot limited to stem cells, pancreatic islet cells, nerve cells, etc.

The present invention will be further illustrated by way of thefollowing Examples. These Examples are non-limiting and do not restrictthe scope of the invention. Unless stated otherwise, all percentages,parts, etc. presented in the Examples are by weight.

EXAMPLES Examples 1A and 1B. Impact of 25HC3S Administration in MiceSubjected to a High Dose of Acetaminophen (ATMP) Example 1A

Materials and Methods

Female mice were peritoneally-injected with acetaminophen (500 mg/kg, in10% ethanol in PBS) either 0.5 or 2 hrs before being treated byadministration of 25HC3S (20 or 25 mg/kg, 10% propylene glycol in PBS).Sera were collected 24 or 48 hrs following acetaminophen administrationand enzymatic activities and other serum parameters were measured.Normal values were obtained from 10 mice who did not receive anyinjection, control mice received only acetaminophen (ATMP) plus vehicle,and experimental mice received ATMP plus 25HC3S.

Results

The results showed that ATMP administration significantly damaged livertissues, increasing serum ALT activities 6-fold; and AST and LDH20-fold. As shown in FIGS. 1A-D, treatment with 25HC3S 2 hours afterATMP administration decreased LDH by 60%; ALT by 58%; and AST by 45%within 24 hrs. In addition, treatment with 25HC3S 0.5 hours after ATMPadministration returned most markers of liver and kidney function tonormal levels in 48 hrs (FIG. 2), whereas untreated control valuesremained elevated.

FIGS. 3A and 3B show the level of 25HC3S in blood (3A) and the indicatedtissues (3B) at the indicated times after administration. As can beseen, the half life of 25HC3S in circulation is about 30 hrs, and thecompound is widely distributed in different tissues in the body.

Example 1B

Materials and Methods

In another set of experiments, female mice were peritoneally-injectedwith acetaminophen (600 mg/kg, in 20% EtOH in PBS) and were then furthertreated by peritoneal injection of 25HC3S (25 mg/kg in 10% PG in PBS) 2and 24 hrs later. Control mice received ATMP without 25HC3S. Mortalitywas monitored for 10 days.

Results

As seen in FIG. 4, the survival rate of animals treated with 25HC3S wasmuch higher than that of control animals, indicating that 25HC3Sprotected the animals from death due to high levels of ATMP.

FIGS. 5A-D show the values for the liver function (damage) markers ALT,AST, AKP and ADH for individual surviving mice. The mice are grouped aseither controls who received ATMP and vehicle (“CON”) or mice whoreceived ATMP and 25HC3S. Sera were sampled at 48 hours. As can be seen,animals to whom 25HC3S was administered in general tended to have lowervalues of each enzyme (i.e. values nearer normal) than control animalswho did not receive 25HC3S. (Note that FIG. 5 does not include data fromanimals that died before sampling.)

Summary of Examples 1A and 1B

Treatment with 25HC3S significantly decreased serum levels of ALT, AST,and ADH activity in mice who received ATMP, and subsequentlysubstantially decreased the mortality of mice who received ATMP. Theseobservations are consistent with efficacy of 25HC3S in protecting theanimals from liver damage caused by high levels of ATMP, and indicatethat 25HC3S can be used as biomedicine for therapy of acute liverfailure induced by ATMP.

Example 2. Impact of 25HC3S on Serum Chemistry Values in Mice Subjectedto a High Dose of Acetaminophen

Materials and Methods.

Mice were challenged with acetaminophen (300 mg/kg PO) and then treatedwith vehicle or 25HC3S (25 mg/kg) by IP injection or PO gavage, one (1)hour and 24 hours after acetaminophen challenge. Measurements andsamples included whole blood for serum and clinical chemistry analysis(ALT, AST, ALK, LDH, BUN and glucose) and livers and kidneys forformalin fixation and histology.

Results

Among acetaminophen-challenged groups, it was noted that groups treatedwith PO vehicle exhibited higher clinical chemistry values when comparedwith groups treated with the IP vehicle and this was also true forgroups that received 25HC3S. Similarly and in general support of this,body weight loss was greater in PO-treated groups vs. IP-treated groups.

Challenge with acetaminophen resulted in body weight loss and markedlyelevated clinical chemistry parameters (LDH, AST and ALT) that peaked at24 hours post-challenge and were returning to normal at 48 hours. When25HC3S-treated groups were compared with the appropriate administrationroute vehicle controls, there was no statistically significantattenuation of body weight loss or elevated clinical chemistryparameters.

In conclusion and under the conditions tested, one (1) hourpost-treatment of acetaminophen challenged mice with 25HC3S (25 mg/kg,IP or PO) did not significantly alter serum chemistry values followingacute liver failure, as induced by PO administration of acetaminophen at300 mg/kg.

Example 3. Kidney Ischemia-Reperfusion

Materials and Methods

Formulation Preparation Procedure:

Preparation of the Formulation for IP Injection:

25HC3S was dissolved in propylene glycol, at 20 mg/mL, and stored atroom temperature as the stock solution. Before use, 3 parts of PBS wasmixed with 1 part of DV928 stock solution. The final concentration forinjection was 5 mg/ml.

Preparation of the Formulation for Oral Gavage:

25HC3S was suspended in 0.5% carboxymethylcellulose (CMC) containing0.05% tween-80, at 10 mg/mL, and stored at room temperature and mixedwell before use.

Methods

Animals:

Adult (9- to 11-week-old) male Lewis (LEW, RE11) rats, 225-250 gram,were housed in a controlled 12-hr light-dark cycle environment andallowed free access to water and regular rat chow. All rats wereanesthetized with pentobarbital 40 mg/kg i.p. Ischemia of the leftkidney was performed by transient occlusion of the left renal artery andvein, and ureter for 50 min with a vascular micro-clip. The skin wastemporarily closed during the ischemia period, and the rats were put ona heating pad maintained at a temperature of 37° C. At reperfusion, theright kidney was removed before closing the abdomen with 4-0 silksuture.

Experiment Design

Animals were randomly divided into 5 groups:

Group A. 25HC3S—i.p. dosing (as pre-treatment) N=12

Group B. 25HC3S—i.p. dosing (as post-treatment) N=12

Group C. 25HC3S—oral dosing (as pre-treatment) N=12

Group D. Vehicle control, i.p. (as pre-treatment) N=6

Group E. Vehicle control, oral (as pre-treatment) N=6

All animals received either active (25HC3S) or vehicle once daily forthe designated period of time as indicated in FIG. 11. Rats in thepre-treatment groups received active or vehicle 1 day before (Day −1)the surgical intervention (I/R event) and continued for a total of 4days. Rats in the post-treatment group received the 1^(st) treatment at30 min after the renal artery occlusion and continued for a total of 3days. Blood samples were taken from all rats 2 days before the surgery(as baseline), 3 days after the surgery, and 7 days after the surgeryfor serum creatinine and blood urea nitrogen (BUN) analysis. All ratswere sacrificed on Day 7.

Results

The results are presented in FIGS. 6A and B. As can be seen,pretreatment by IP injection of 25HC3S a day before the surgerysignificantly improved rat serum creatinine and BUN levels.Post-treatment by IP injection also reduced rat serum creatinine and BUNlevels. Administration of 25HC3S by oral gavage, although given a daybefore the surgery and at a higher dose, reduced rat serum creatinineand BUN levels on Day 3, but to a lesser extent than administration byinjection.

Example 4. Heart Ischemia-Reperfusion

Materials and Methods

Wild-type C67B16 mice, both males and females, were used in theexperiment. After anesthesia, the thorax of each mouse was opened andthe heart was subject to a 45-minute ischemic period by ligation of theleft anterior descending coronary artery, and then reperfusion wasallowed by removing the coronary arterial obstruction. The thorax wasclosed, and mice were allowed to recover for 24 hours. All animals weresacrificed at the end of the study, 24 h after the procedure, to gainaccess to the heart tissue. The heart was frozen, sectioned into slices,and then stained to determine the infarct size.

Given the experience that 25-30% of the animals may die within the24-hour period because of the procedure (occlusion of coronary artery),all groups included 12 animals. The vehicle treated group (12 mice) wascompared with 25HC3S treated groups (24 mice). One group of micereceived the drug (25HC3S) just before ischemia/reperfusion (PR) andanother group received drug about 16-20 h before FR. Administration wasvia the i.p. route with either vehicle (10% propylene glycol in PBS) or25HC3S at a dose of 25 mg/kg in the same vehicle.

Results

The data showed no statistically significant difference in sizes ofinfarction when comparing vehicle and drug treated mice. However, the24-hour survival rate in the vehicle treated group was 64% vs. 86% inthe 25HC3S treated group (FIG. 7), indicating that administration of25HC3S reduces mortality after heart ischemia-reperfusion injury.

Example 5. Brain Stroke Study in Rats

Methods and Materials

Transient focal cerebral ischemia was produced by right side middlecerebral artery (MCA) occlusion in male Sprague Dawley rats underanesthesia. After 120 min of ischemia, the MCA blood flow was restored.The procedure was therefore named tMCAO. All rats were given an i.p.injection of saline (4 ml per rat) after the surgery. Three groups ofrats were used in the study:

Group A: 12 sham operated rats received vehicle by ip injection

Group B: 12 tMCAO rats received vehicle by ip injection

Group C: 12 tMCAO rats received 25HC3S by ip injection

The parameters measured were as follows:

Mean body weight over time

Mean 7- and 20-point Neuroscore over time

Limb Placing test results over time

Mean infarct volumes (mm3), edema volumes (mm3) and T2-relaxation times(ms)

Behavioral testing was conducted at 24 hours, 3 days, and 7 days posttransient middle cerebral artery occlusion (tMCAO). Both 20-pointNeuroscore and 7-point Neuroscore tests were conducted to assesspost-ischemic motor and behavioral deficits. The Limb Placing test wasconducted to assess the sensory motor integration of fore- andhind-limbs responses to tactile and proprioceptive stimulation.

MRI acquisitions in vivo for all rats were performed at 24 h and 7 daysafter tMCAO. Lesion size, tissue viability (T2 in milliseconds), andbrain edema were determined using absolute T2-MRI. Eighteen (18) coronalslices of thickness 1 mm were acquired using field-of-view imagingmatrix. Absolute T2-values from contra-lateral cortex were used as areference for tissue viability. All MRI data were analyzed using Matlabsoftware. The infarct volume/oedema analysis was done by an observerblinded to the treatment groups.Results

The surgery of tMCAO typically introduces 20-25% mortality in animals.In this study, two animals in the treatment group died immediately afterthe brain surgery. One animal in the sham group, one animal in thevehicle group, and two animals in the treatment group died during theMRI measurement at 24 hour after the surgery. One animal from thevehicle group and one animal from the treatment group died 2 days or 3days after the surgery. In addition, one animal in the vehicle group didnot show any sign of tMCAO injury by all three behavioral tests, MRImeasurements (criteria for exclusion), or body weight changes,indicating no surgical occlusion happened. The data from this animal wasexcluded from analysis. Another animal in the vehicle group also showedminimal sign of tMCAO injury by all three behavioral tests and tissueviability (T2 in ms), while no sign of injury by lesion volume and brainedema (same values as sham). Its body weight did not drop as did that ofall other animals but increased after the surgery. However, the datafrom this animal was included in all analysis.

In this study, tMCAO characteristically induced both functional deficits(by behavioral tests) and brain pathology (by MRI) in all animals.Although there was a consistent trend of better scores in every test andevery time point in animals receiving 25HC3S than those receivingvehicle, there were no statistically significant differences betweenvehicle group and treated group in 7-point Neuroscore (FIG. 8A),20-point Neuroscore (FIG. 8B), or Limb Placing (FIG. 8C) test. A trendof recovery over time post tMCAO among all 3 behavioral tests was alsonoticeable in all animals. The lack of differences, if not a bettertrend, in treated animals relative to vehicle group in all 3 behavioraltests indicated that the treatment with 25HC3S did not cause irritation,discomfort, or any adverse side effects in these animals.

Brain edema is an acute response after ischemia/reperfusion injury,which typically peaks within 3 days after tMCAO and mostly recovers at 7days after the procedure. In this study, all animals showedcharacteristic brain edema after tMCAO. Animals receiving 25HC3S showedsmaller edema volume (FIG. 8F) or % edema (FIG. 8H) at 24 hours posttMCAO as compared to animals in the vehicle group, although thedifferences were not statistically significant. At 7 days post tMCAO,the brain edema, either edema volume or % edema, in both vehicle andtreated groups were nearly recovered (FIGS. 8G and 8I).

Animals treated with 25HC3S appeared to have smaller brain lesionvolumes than those receiving the vehicle at both 24 hours (FIG. 8D) and7 days (FIG. 8E) after tMCAO, although the differences were notstatistically significant. The lesion volume tended to decrease orrecover in both groups over time, comparing 24 hours with 7 days posttMCAO.

Animals treated with 25HC3S showed statistically higher tissueviability, as expressed by lower T2 relaxation time in ms, than thoseanimals receiving the vehicle. This higher brain tissue viability (orlower T2 in ms) in treated animals than that in vehicle group wasapparent at both 24 hours (FIG. 8J) and 7 days (FIG. 8K) post tMCAO. Atrend of recovery was seen in both groups.

A significant but expected body weight drop was observed in all animalsreceiving the tMCAO procedure (FIG. 8L), although the body weights inthe treatment group were decreased one day after the dosing or beforethe tMCAO procedure. However, consistent with other differences,regardless of statistical significance, including trends seen in brainpathology and behavioral tests, animals receiving 25HC3S showed a fasterrecovery in body weights than the vehicle animals receiving vehiclestarting from Day 4 after the procedure.

Summary

The results suggest a beneficial effect of 25HC3S in this rat tMCAOmodel. For instance, the MRI results indicate that 25HC3S appeared toprotect the brain from acute ischemia injuries. At 24 hr after thesurgery, 25HC3S treated rats showed smaller brain lesion volume andstatistically significantly smaller T2 lesion. The brain edema values(both edema volume and % edema) tended to be less than those rats in thevehicle group. Although some higher levels of edema (both edema volumeand % edema) were found in 25HC3S treated rats at 7 days after thesurgery, the lesion volume and T2 lesion were again smaller in 25HC3Streated rats than in rats receiving vehicle. In particular, thetreatment resulted in statistically significantly improved brain T2lesion values in 25HC3S treated rats, compared to untreated rats, atboth 24 hr and 7 days after the surgery.

Examples 6A-C. Sepsis Examples 6A and 6B

Materials and Methods

To examine the effect of 25HC3S on sepsis induced by the endotoxinlipopolysaccaride (LPS), 11-week-old female C57BL/6J mice wereIV-injected with LPS (30 mg/kg or 40 mg/kg, in PBS) 2 hrs before beingtreated by administration of 25HC3S (25 mg/kg, 10% propylene glycol inPBS), and mortality was monitored (′)/0 survival).

Results

Data from these two experiments is depicted in FIGS. 9A and B. As can beseen, in both experiments, mice that received 25HC3S lived significantlylonger than mice that received only vehicle.

Example 6C

Materials and Methods

A further experiment to examine the effects of pretreatment of 25HC3S onmortality induced by LPS was conducted. In this experiment, 11-week-oldfemale C57BL/6J mice were IV-injected with LPS (4 mg/kg in PBS). Themice were treated by administration of 25HC3S (50 mg/kg, 10% propyleneglycol in PBS) 2 hrs before LPS administration, and mortality (%survival) was monitored. The results are presented below and in FIG. 9C.As can be seen:

Results

Control Group:

At day 1, one of five (1/5) mice died, i.e. the survival rate was 80%;

By day two, two of five (2/5) mice had died, i.e. the survival rate was60%;

By day 3, three of five (3/5) mice had died, i.e. the survival rate was40%.

25HC3S Treated Group:

As of day 3, none of the five mice treated with 25HC3S had died, i.e.the survival rate was 100%.

SUMMARY

Administration of 25HC3S prolongs life in mice exposed to LPS.

Example 7. Human Phase I Single Dose Ascending Study

A randomized, double blind, and placebo controlled single dose ascendingFirst-in-human Phase 1 study was conducted. The active agent, 25HC3S,was suspended at 30, 100, 300, and 600 mg in 60 mL of ORA-Blend® SFsugar-free flavored oral suspending vehicle. As Placebo dosage form,calcium carbonate, USP, was suspended at 30, 100, 300, and 600 mg in 60mL of ORA-Blend® SF sugar-free flavored oral suspending vehicle. Thesuspension, either active or Placebo, was orally administered to eachsubject. Each dose group had 4 subjects receiving a single dose ofactive and 2 subjects receiving a single dose of Placebo. All subjectswere monitored by health professionals for any potential adverse eventsfor 7 days following administration. Plasma samples were collected atselected time intervals before and after the administration, asindicated in FIG. 10.

No adverse effects were observed in any dose group, including those whoreceived 600 mg of 25HC3S. Both active and placebo were well tolerated.Pharmacokinetically, the drug exhibited high bioavailability (FIG. 10).

Unless otherwise stated, a reference to a compound or component includesthe compound or component by itself, as well as in combination withother compounds or components, such as mixtures of compounds.

As used herein, the singular forms “a,” “an,” and “the” include theplural reference unless the context clearly dictates otherwise.

For all numeric ranges provided herein, it should be understood that theranges include all integers between the highest and lowest value of therange, as well as all decimal fractions lying between those values, e.g.in increments of 0.1.

For all numeric values provided herein, the value is intended toencompass all statistically significant values surrounding the numericvalue.

While the invention has been described in terms of its preferredembodiments, those skilled in the art will recognize that the inventioncan be practiced with modification within the spirit and scope of theappended aspects and claims. Accordingly, the present invention shouldnot be limited to the embodiments as described above, but should furtherinclude all modifications and equivalents thereof within the spirit andscope of the description provided herein.

The invention claimed is:
 1. A method of treating acute kidneydysfunction or acute kidney failure in a subject in need thereof,comprising administering to the subject an amount of5-cholesten-3,25-diol, 3-sulfate (25HC3S) or pharmaceutically acceptablesalt thereof that is sufficient to treat the acute kidney dysfunction oracute kidney failure, wherein the subject does not have hyperlipidemia.2. The method of claim 1, wherein the 25HC3S or pharmaceuticallyacceptable salt thereof is administered at a dose ranging from about0.001 mg/kg to about 100 mg/kg.
 3. The method of claim 1, wherein theadministering is performed orally or by injection.
 4. The method ofclaim 1, wherein the administering is performed orally.
 5. The method ofclaim 1, wherein the administering is performed by injection.
 6. Themethod of claim 1, wherein the administering is performed orally,parenterally, intravenously, intramuscularly, subcutaneously, byintradermal injection, by intraperitoneal injection, transdermally,sublingually, rectally, by buccal delivery, by inhalation of an aerosol,intravaginally, intranasally, or topically.
 7. The method of claim 1,wherein the 25HC3S or pharmaceutically acceptable salt thereof isadministered in a formulation further comprising a pharmaceuticallyacceptable carrier.
 8. The method of claim 1, wherein the 25HC3S orpharmaceutically acceptable salt thereof comprises a pharmaceuticallyacceptable salt of 25HC3S.
 9. The method of claim 2, wherein the 25HC3Sor pharmaceutically acceptable salt thereof comprises a pharmaceuticallyacceptable salt of 25HC3S.
 10. The method of claim 3, wherein the 25HC3Sor pharmaceutically acceptable salt thereof comprises a pharmaceuticallyacceptable salt of 25HC3S.
 11. The method of claim 4, wherein the 25HC3Sor pharmaceutically acceptable salt thereof comprises a pharmaceuticallyacceptable salt of 25HC3S.
 12. The method of claim 5, wherein the 25HC3Sor pharmaceutically acceptable salt thereof comprises a pharmaceuticallyacceptable salt of 25HC3S.
 13. The method of claim 6, wherein the 25HC3Sor pharmaceutically acceptable salt thereof comprises a pharmaceuticallyacceptable salt of 25HC3S.
 14. The method of claim 7, wherein the 25HC3Sor pharmaceutically acceptable salt thereof comprises a pharmaceuticallyacceptable salt of 25HC3S.
 15. The method of claim 4, wherein the 25HC3Sor pharmaceutically acceptable salt thereof is administered at a doseranging from about 0.001 mg/kg to about 100 mg/kg.
 16. The method ofclaim 5, wherein the 25HC3S or pharmaceutically acceptable salt thereofis administered at a dose ranging from about 0.001 mg/kg to about 100mg/kg.
 17. The method of claim 1, wherein the 25HC3S or pharmaceuticallyacceptable salt thereof is administered at a dose ranging from about 0.1mg/kg to about 10 mg/kg.
 18. The method of claim 1, wherein theadministering occurs for not more than 14 days.